Novel Secondary Metabolites from Endophytic Marine-Derived Fungi

Novel Secondary Metabolites from Endophytic Marine-derived Fungi

Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn

vorgelegt von Celso Almeida aus Lissabon, Portugal

Bonn 2011

Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn

1. Gutachterin : Prof. Dr. G. M. König 2. Gutachterin : Prof. Dr. E. Kostenis

Tag der Promotion : 15. Februar 2011 Erscheinungsjahr : 2011

Vorveröffentlichungen der Dissertation/In Advance Publications of the Dissertation

Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Mathematisch- Naturwissenschaftlichen Fakultät, vertreten durch die Mentorin/Betreuerin der Arbeit, in folgenden Beiträgen vorab veröffentlicht:

Parts of this study have been published in advance by permission of the Mathematisch- Naturwissenschaftlichen Fakultät, represented by the supervisor of this study:

Publikationen / Research Papers

Almeida C., Eguereva E., Kehraus S., Siering C. and König G.M., Hydroxylated Sclerosporin Derivatives from the Marine-derived Fungus Cadophora malorum, J. Nat. Prod., 2010, 73 (3), pp 476–478.

Almeida C., Elsaedi S., Kehraus S. and König G. M., Novel Bisabolane Sesquiterpenes from the Marine-derived Fungus Verticillium tenerum, Nat. Prod. Commun. 2010, 5(4), 507- 10.

Almeida C., Part N., Kehraus S. and König G. M., Stachylines A – D from the Sponge-derived Fungus Stachylidium sp. (accepted by Journal of Natural Products).

Almeida C., Kehraus S., Prudêncio M. and König G. M., Marilones A – J, Unusual Phthalides from the Sponge-derived Fungus Stachylidium sp. (submitted to European Journal of Organic Chemistry).

Almeida C., Kehraus S., Dimas K., Gütschow M. and König G. M., Marilines A – D, Novel Phthalimidines from the Sponge-derived Fungus Stachylidium sp. (In preparation)

Almeida C., Kehraus S. and König G. M., Novel Furyl Derivative from the Sponge-derived Fungus Stachylidium sp. (In preparation).

Almeida C., Kehraus S. and König G. M. Novel bioactive peptides from the Sponge-derived Fungus Stachylidium sp. (In preparation). 4

Tagungsbeiträge/Research Presentations

Almeida C., Kehraus S., König G. M., Hydroxylated derivatives of a rare cadinane-type sesquiterpene isolated from a cytotoxic Wardomyces inflatus extract. Poster presented at the 7th Natural Products Joint Meeting of AFERP, ASP, GA, PSE & SIF, August 3-8, 2008, Athens, Greece.

Almeida C., Kehraus S., König G. M., Hydroxylated derivatives of a rare cadinane-type sesquiterpene isolated from a cytotoxic Wardomyces inflatus extract. Poster presented at the Joint Meeting of the Deutsche Pharmazeutische Gesellschaft (DPhG), October 8-11, 2008, Bonn, Germany.

Almeida C., König G. M., Novel secondary metabolites from the sponge-derived fungus Stachylidium sp.; tracking bacterial endosymbionts. Oral presentation at the 50th Annual Meeting of the American Society of Pharmacognosy, June 27-July 1, 2009, Honolulu, Hawaii.

Almeida C., König G. M., Novel Secondary Metabolites from the Sponge-Derived Fungus Stachylidium sp.; Tracking Bacterial Endosymbionts. Poster presented at the 6th European Conference on Marine Natural Products, July 19 - 23, 2009, Porto, Portugal.

Almeida C., Kehraus S., Dimas K., and König G. M., Marilines A – D, Novel Phthalimidines from the Sponge-derived Fungus Stachylidium sp. Poster presented at the International Conference New Biotrends in Green Chemistry, December 1-2, 2010, Dortmund, Germany.

To my mother, Marilia, and father, Benjamim

Acknowledgements I wish to express my sincere gratitude and admiration to my supervisor Prof. Dr. G. M. König for the expert guidance, encouragement and kind support during the course of this project. I would like to thank her for providing excellent scientific advice, working facilities, for letting me express my creativity, and especially for understanding and guidance in unexpected personal difficulties that arose during the research. I have nothing else than to be proud and consider to be a truly privilege to have performed a PhD under the orientation of Prof. Dr. G. M. König.

Special thanks go to Prof. Dr. E. Kostenis for officiating as second referee.

Many specific tasks involved in this study were performed in cooperation with other members of the Institute for Pharmaceutical Biology, Uni Bonn. For this work cordial thanks go to: Dr. Stefan Kehraus for indispensable help concerning the implementation and interpretation of special NMR experiments, for proofreading manuscripts and thesis, and for providing friendly, professional laboratory support during all phases of this study. I would also like to express my extreme admiration for the NMR interpretation skills of Dr. Kehraus. Ekaterina Eguereva for introducing me to the work with marine-derived fungi. Also thanks for recording all LC-MS spectra and friendly talks during the research. Edith Neu for conducting agar diffusion assays and for being such a friendly person. Natalja Part, for her outstanding lab work and for being a good friend. E. Gassen for friendly assistance and for resolving all administrative questions. I would like to thank all members of the Institute for Pharmaceutical Biology, University of Bonn, present or past for cooperation and friendship, with special regards to Hendrik Greve and Ana Kralj for the great help in the first year in the lab.

Extra-special thanks go to Sanem Insu Tezkan, words cannot describe her existance. As for Rita Roque, Rui Branco and Paulo Figueiredo, thank you for making the days in Germany so special, I met you here but the friendship will be for life. Also for Maria Laura Vinuela which will be in my heart for the rest of my life. Another thanks goes to Ricardo Baptista Lopes, long time friend that is allways there when i return to Portugal. A final thankfull word for “pre-PhD“ personalities, namely Dr. Rob Verpoorte (Leiden University, The Netherlands) for all the knowledge given during my Masters, and Dr. Artur Silva

(Aveiro University, Portugal) for teaching exquisidly the basics of NMR spectroscopy to a biologist, which was essencial for this PhD work.

Financial support for this project was provided by FCT (Fundação para a Ciencia e Tecnologia, Portugal) and is gratefully acknowledged.

International scientific cooperation acknowledgements. I thank the efforts of Dr. K. Dimas (Biomedical Research Foundation of Academy of Athens, Greece) for the cytotoxicity assays, Prof. Dr. M. Gütschow (Institute for Pharmaceutical Chemistry, University of Bonn, Germany) for performing the panel of proteases inhibition assays, Dr. L. Meijer (Protein Phosphorylation & Disease, CNRS, Roscoff, France) for performing the protein kinases assays and Dr. C. Pannecouque (Rega Institute for Medical Research, Leuven, Belgium) for performing the HIV-1 and HIV-2 antiviral assays; I also kindly thank Dr. M. Prudêncio (Malaria Unit, Institute for Molecular Medicine, University of Lisbon, Portugal) for performing the antiplasmodial activity assays, Indra Bergval (KIT Biomedical Research, Royal Tropical Institute, Amsterdam, Netherlands) for performing the M. tuberculosis activity assays, Dr. Marc Diederich (LBMCC, Luxembourg) for performing the NF-kB activity assays, Dr. Steinar Paulsen (University of Tromsø, MabCent, Tromsø, Norway) for performing the anti-diabetic activity assays and Dr. K. Shimokawa (Department of Chemistry, Nagoya University, Japan) for performing the 3T3-L1 murine adipocytes assay; the Ki determinations and antagonist functional data that was generously provided by the National Institute of Mental Health's Psychoactive Drug Screening Program, Contract # HHSN-271-2008-00025-C (NIMH PDSP). The NIMH PDSP is directed by Bryan L. Roth MD, PhD at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscol at NIMH, Bethesda MD, USA; I kindly thank also the remaining antiviral tests performed by the U.S. National Institute of Health which were supported by contracts NO1-A1-30048 (Institute for Antiviral Research, IAR) and NO1-AI-15435 (IAR) from Virology Branch, National Institute of Allergic and Infectious Diseases, NIAID; also a thankful word for Dr. M. Engeser and Dr. B. Sondag (Kekulé-Institute for Organic Chemistry and Biochemistry, University of Bonn) for the High Resolution Mass Spectrometry efforts. Synthetic reference compounds for CD spectra comparison with verticonols A/B were kindly provided by Dr. Kenji Mori, Emeritus Professor from the University of Tokyo, Japan; special thanks to Carsten Siering (Kekulé Institute for Organic and Inorganic Chemistry, University of Bonn) for CD spectral measurements and invaluable discussions.

Table of contents Page 1. Introduction …………………………….…………………………………….….1

2. Scope of the present study …………………………………….………...……..14

3. General methodology ……………………………………………….…………15

4. Marilines A – D, novel phthalimidines from the sponge-derived

fungus Stachylidium sp...... 18

5. Marilones A – J, unusual phthalides from the sponge-derived

fungus Stachylidium sp...... 36

6. Stachylines A – D from the sponge-derived fungus Stachylidium sp...... 56

7. Novel furyl derivative from the sponge-derived fungus Stachylidium sp...... 70

8. Endolides A – J, N-methylated peptides from the sponge-derived

fungus Stachylidium sp...... 75

9. Hydroxylated sclerosporin derivatives from the marine-derived

fungus Cadophora malorum …………………………………….……….…….76

10. Novel bisabolane sesquiterpenes from the marine-derived Fungus

Verticillium tenerum ...... 84

11. Discussion ……………………………………………………………..….……92

12. Summary …………………………………………………………..…..……….96

13. Appendix

13.1. Bioactivity results ……………………………………..…..……………109

13.2. Protocols and media ………………………………...…………………..121

13.3. Complete 2D NMR data ………………………….………...…………..127

13.4. Spectroscopic data supporting information ………………....………….147

13.5. 1H and 13C NMR spectra

13.5.1. 1H and 13C NMR spectra of new molecules ……………….....160

13.5.2. 1H and 13C NMR spectra of known molecules …………...…..185 9

Abbreviations

°C degrees Celsius 1D one dimensional 2D two dimensional T [α] D specific rotary power, sodium D-line (589 nm); T: temperature δ NMR chemical shift [ppm] λ wavelength [nm] μ micro (10-6) μg 10-6 gram μl 10-6 liter μM 10-6 molar, micromolar (= 10-6 mol/L) ν  wave number [cm-1] ASW artificial seawater ATR attenuated total reflection AU absorbance units BMS biomalt salt medium br broad (in connection with NMR data) c concentration C 18 C-18 modified silica gel calcd calculated CD circular dichroism

CDCl3 chloroform-d

CD3CN acetonitrile-d3

CD3OD methanol-d4

CH2Cl2 dichloromethane (DCM)

CH3CN acetonitrile conc. concentration COSY correlated spectroscopy cm 10-2 meter CZ Czapek medium d doublet (in connection with NMR data) DAD diode array detector 10

DCM dichloromethane DEPT distortionless enhancement by polarization transfer dmol 10-1 mol DNA deoxyribonucleic acid

EC50 half maximal effective concentration (drug concentration causing 50% of maximal effect) e.g. example given (for example) EI electron ionization ESI electron spray ionization et al. et alii [Lat.]: and others EtOAc ethyl acetate EtOH ethanol g gram GI growth inhibition h hour

H3BO3 boric acid HMBC heteronuclear multiple-bond correlation HMGCoA hydroxymethylglutaryl-CoA HPLC high performance liquid chromatography HR high resolution HSQC heteronuclear single quantum correlation Hz Hertz

H2O water

IC50 half maximal inhibitory concentration (drug concentration causing 50% inhibition) i.e. that is IR infrared J spin-spin coupling constant [Hz] kDa kilo Dalton (= 103 Dalton) L liter LC liquid chromatography leu leucine m meter

m multiplet (in connection with NMR data) m/z mass-to-charge ratio (in connection with mass spectrometry) mdeg millidegrees Me methyl MeOH methanol

MeOD methanol-d4 mg 10-3 gram MHz megahertz min minute mL 10-3 liters mm 10-3 meters mM 10-3 molar, millimolar (= 10-3 mol/L) mol. wt. molecular weight [g/mol] MYA malt yeast agar medium MS mass spectrometry NaOH sodium hydroxide n.d. not determined

NH4Ac ammonium acetate nm 10-9 meter NMR nuclear magnetic resonance no number NOE nuclear Overhauser effect NOESY nuclear Overhauser effect spectroscopy NP normal phase silica gel NRPS non-ribosomal peptide synthetase p pentet (in connection with NMR data) PDA photodiode-array PE petroleum ether pH potentia hydrogenii PKS polyketide synthase ppm parts per million q quartet (in connection with NMR data) qC quaternary carbon

ROESY rotating frame Overhauser effect spectroscopy RP reversed phase RT room temperature s singlet (in connection with NMR data) sec second Si silica gel sp. species spp. species (plural) sxt sextet (in connection with NMR data) t triplet (in connection with NMR data) TLC thin layer chromatography UV ultraviolet VIS visible VLC vacuum-liquid chromatography X-ray Röntgen-ray

1. Introduction

Nature has evolved over time to produce a bewildering diversity of secondary metabolites. Based on empirical observations and folklore, natural product extracts were the first, and for a long time, the only medicines available to mankind. Although according to the WHO, 80% of the world’s population - primarily those of developing countries - rely on plant-derived medicines for their healthcare, they are largely supplanted by pharmaceutical ingredients in the Western world. Furthermore, the dependence upon natural products is no longer obligatory and many drugs are purely synthetic small molecules or manufactured biologics such as vaccines, antibodies, and recombinant proteins (Gurib-Fakim, 2006; Ganesan, 2008).

1.1. Role of Natural Products in Pharmaceutical Sciences

The seminal discoveries leading to the use of pure drug substances occurred in the 18th and 19th centuries. In 1785 William Withering published his treatment of heart patients with cardiotonic foxglove extract, also known as digitalis (Aronson, 1985). This treatment led to the discovery of digoxin (Smith, 1930), and has been used to treat arrhythmia and congestive heart failure. In 1806 Freidrich Serturner (Schmitz, 1985) analyzed opium poppy and isolated morphine, used in pain therapy. The pharmacology of the small molecule morphine enabled an understanding of the opiate receptors subtypes, of the endorphin and enkephalin pathways (Ignelzi, 1980), and ultimately led to the development of analogs as therapy for pain. In 1897 Felix Hoffmann, working with the Bayer Company, synthesized aspirin from salicylic acid in willow bark (Bosch and Banos, 1998). People had been using the extract from willow bark prepared as a tea for centuries to treat rheumatism and headache. After structure optimization with an acetylation reaction to mask the phenol of salicylic acid, Aspirin was born. Using salicylic acid as a pharmacologic tool, it was possible to delineate the mechanisms of inflammation (Vane, 1971) and subsequently to design and test a battery of new nonsteroidal anti-inflammatory agents, including acetaminophen, ibuprofen, and naproxen. Although mycophenolic acid was the first fungal secondary metabolite obtained from Penicillium glaucoma as early as 1896 (see Bérdy, 2005), it is a well- known story that in 1928, Alexander Fleming discovered penicillin in mold (Bennett

and Chung, 2001). The discovery of penicillin and its impact on the treatment and understanding of infectious disease did more for human health than any other single discovery. The small molecule penicillin enabled the study of antibiotic action and infection to the point that countless penicillin and cephalosporin could be synthesized and studied for the treatment of various strains of bacteria and in response to antibiotic resistance (Ghuysen 1997). An understanding of infectious disease was achieved, and an entirely new therapeutic approach was born. OH O HO O O O

H mycophenolic acid R2 H N S R H N O S N O O R1 N O O OH CH2OOH

core structure of penicillins core structure of cephalosporins

1.2. Natural products as pharmaceutical drugs in the 20th century

Natural products continued to play a major role, and endogenous chemicals, such as the steroids, prostaglandins, and peptide hormones, provided the pharmaceutical industry with additional natural inspiration for drug discovery during the 20th century (Gaudilliere, 2005). The most recent survey covering the period 1981–June 2006 of molecules discovered post-1970 lists a total of 1184 new chemical entities (NCEs) receiving approval as pharmaceutical drugs (Newman and Cragg, 2007). Of these, 52% have a natural product connection, 18% are biologics, and 30% purely synthetic. The meta-analysis of this list made by Ganesan (2008) reveals a total of 24 unique natural product chemotypes that led at least to one approved drug (Table 1). A priori, natural products should undergo an iterative cycle of pharmacological improvement, as their evolutionary reason for existence is not for use as a therapeutic agent (Ganesan, 2008). Though, among the 24 leads in Table 1, 17 progressed to an approved drug with no modification. Another six natural products were modified by semisynthesis and only the β-lactam SQ26,180 was replaced by a synthetic analog.

Table 1. The 24 natural products discovered since 1970 that led to an approved drug in 1981–2006, (Ganesan, 2008) Lead, year Chemical class Origin Drug, year Rank* Validamycin, 1970 Oligosaccharide Actinomycete Acarbose, 1990 357 Voglibose, 1994 Midecamycin, 1971 Macrolide Actinomycete Miocamycin, 1995 Pseudomonic acid, 1971 Polyketide Bacteria Mupirocin, 1985 436 Taxol, 1971 Diterpene Plant Paclitaxel, 1993 81 Docetaxel, 1995 123 Cephamycin C, 1971 β-lactam Actinomycete Moxalactam, 1982 Cefotetan, 1984 Cefbuperazone, 1985 Coformycin, 1974 Nucleoside Actinomycete Pentostatin, 1992 Echinocandin B, 1974 Cyclopeptide Fungus Caspofungin, 2001 293 Micafungin, 2002 Anidulafungin, 2006 Mizoribine, 1974 Nucleoside Fungus Mizoribine, 1984 Rapamycin, 1974 Polyketide Actinomycete Sirolimus, 1999 434 Everolimus, 2004 Zotarolimus, 2005 Compactin, 1975 Polyketide Fungus Lovastatin, 1984 264 Simvastatin, 1988 2 Pravastatin, 1989 41 Fluvastatin, 1994 195 Atorvastatin, 1997 1 Cerivastatin, 1997 Pitavastatin, 2003 71 Rosuvastatin, 2003 Cyclosporine A, 1975 Cyclopeptide Fungus Ciclosporin, 1983 122 Lipstatin, 1975 Polyketide Actinomycete Orlistat, 1987 277 Bestatin, 1976 Peptide Actinomycete Ubenimex, 1987 Thienamycin, 1976 β-lactam Actinomycete Imipenem, 1985 247 Meropenem, 1994 231 Panipenem, 1994 Faropenem, 1997 Biapenem, 2002 Ertapenem, 2002 Doripenem, 2005 Artemisinin, 1977 Sesquiterpene Plant Artemisinin, 1987 Artemether, 1987 Artenusate, 1987 Artheether, 2000 Forskolin, 1977 Diterpene Plant Colforsin, 1999 Plaunotol, 1977 Diterpene Plant Plaunotol, 1987 Avermectib B1a, 1979 Polyketide Actinomycete Ivermectin, 1987 SQ26,180, 1981 β-lactam Actinomycete Aztreonam, 1984 Carumonam, 1988 Spergualin, 1981 Peptide Bacteria Gusperimus, 1994 Arglabin, 1982 Sesquiterpene Plant Arglabin, 1999 FK506, 1984 Polyketide Actinomycete Tacrolimus, 1993 103 Daptomycin, 1986 Cyclodepsipeptide Actinomycete Daptomycin, 2003 Calicheamicin γ1, 1988 Polyketide Actinomycete Gemtuzumab, 2000 *final column gives the ranking among the global top 500 drugs of 2006, according to IMS Health.

Although leadfinding new bioactive carbon skeletons is essential for the development of new drugs, known successful lead compounds continue to give us optimized pharmaceutically approved drugs or currently in clinical trials. Two of these examples are Larotaxel (XRP9881) and Cabazitaxel (XRP6258), novel taxoid compounds that appear to be potent microtubule stabilizers (NCI Drug Dictionary: Larotaxel. http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=42554. Accessed 1 December 2010; Engels et al., 2005). Larotaxel and Cabazitaxel are active in cell lines resistant to doxorubicin, vinblastine, paclitaxel, and docetaxel (Gelmon et al., 2000; Fumoleau et al., 2005). Both compounds appear to cross the blood-brain barrier (Sessa et al., 2002). Furthermore, Larotaxel shows in vivo synergy in combination with doxorubicin, cisplatin, vinorelbine, and trastuzumab (Vrignaud et al., 2005; Vrignaud et al., 2007). Larotaxel is currently in phase III trials in patients with advanced pancreatic cancer and in patients with advanced bladder cancer. Cabazitaxel is also currently in a phase III trial in patients with hormone-refractory prostate cancer following docetaxel- based therapy. (http://www.oncology.sanofi-aventis.com. Accessed 1 December 2010).

O O O OH

O NH O

O OH O O OH O O

taxol (paclitaxel)

O O O O OCH O H3CO 3 O O NH O O NH O H O O H O OH O O O OH OH O O O OH O O O

larotaxel cabazitaxel

1.3. Terrestrial Fungal-derived Natural Products as Pharmaceutical Drugs

A wide range of pharmaceutically significant compounds belonging to all structural classes were found to be produced by fungi (Mutschler et al., 2008). Since the discovery of the fungal metabolite penicillin by Fleming in 1928 and its subsequent development as antibiotic drug, secondary metabolites of fungal origin have been proved to be an important source for new pharmaceuticals and drug lead compounds, as shown by the presently bestselling pharmaceutical drugs, the fungal-derived statins, cholesterol- lowering drugs based on inhibition of HMG-CoA reductase which are widely consumed daily and play an important role in lowering the risk of cardiovascular disease (Laws et al., 2004). Besides the mentioned fungal-derived compounds in Table 1 (compound discovery post-1970), other fungal metabolites that are present on the pharmaceutical market should be mentioned, such as semi-synthetic or synthetic penicillins and cephalosporins, the alkaloid ergotamine (Ergo-Kranit®), the antibiotic polyketide griseofulvin (Likuden M®), the immunosuppressive mixed-biosynthesized compound mycophenolate mofetil (CellCept®, derivative of mycophenolic acid) used for preventing renal transplant rejection as well as the antibacterial terpenoid fusidic acid (Fucidine®) (Hamilton- Miller, 2008; Buttler, 2005), O OO O O O H O O HO O Cl O O H N N griseofulvin N N N O O O O HO cyclosporine A N N H H compactin (mevastatin) N N N N H O O O O O HO OH OHO NH O N N O HO O H HO O H HN HO O O NH O H H OH O H N H HO N echinocandin B

HO fusidicacid H OH H OH O OH 5

A class of antimicrobial agents that remained largely undeveloped for human clinical use is the pleuromutilins (Hunt, 2000). These antimicrobials are derivatives of the naturally occurring pleuromutilin produced by Pleurotus mutilus, an edible mushroom. In veterinary practice, tiamulin and valnemulin (two semisynthetic pleuromutilin analogs) are used for the control and treatment of serious infections in swine (Hunt, 2000, Brooks et al. 2001). Recently, retapamulin, a semisynthetic pleuromutilin compound was approved in the U.S. and Europe for the treatment of skin infections (Daum et al., 2007; Jacobs, 2007; Laustsen et al., 2008). Retapamulin has a unique mode of action, selectively inhibiting the elongation phase of bacterial protein synthesis at a unique site on the prokaryotic ribosome (Hunt, 2000). This antibacterial drug currently shows no target-specific cross-resistance to other classes of antibiotics and has potent activity in vitro against Staphylococcus aureus and Streptococcus pyogenes, including strains that are resistant to ß-lactams, macrolides and quinolones, as well as strains that are resistant to current topical therapies including fusidic acid and mupirocin (Bouchillon et al., 2005; McCloskey et al., 2005; Stevens et al., 2005).

OH OH O O HO N S H O O H

O O

pleuromutilin retapamulin

But to date, fungal metabolites or derivatives thereof have not been launched as anticancer chemotherapeutics. Interestingly, several plant-derived anti-cancer agents are being reported to be produced from endophytic fungi isolated from the original plant sources. Stierle and coworkers have demonstrated in 1993, that the well-established and world’s bestselling anticancer drug taxol from the plant Taxus brevifolia, is also produced by the endophytic fungus Taxomyces andreanae (Stierle et al., 1993). In the meantime, taxol was discovered as product of many various endophytic fungi (Yang et al., 1994; Wang et al. 2000; Ji et al., 2006). Also recently the aryl tetralignan podophyllotoxin has been reported to be produced by endophytes, namely

Phialocephela fortinii, isolated from the rhizomes of the host plant Podophyllum peltatum (Eyberger et al., 2006). Podophyllotoxin, which was originally isolated from the rhizoma of Podophyllum peltatum, is a valuable natural product as precursor for several therapeutic agents, including the anticancer drugs etoposide, teniposide and etoposide phosphate (Canel et al., 2000). In other recent reports, camptothecin was also found to be produced by endophytic fungi (Puri et al., 2005; Strobel & Daisy, 2003; Strobel et al., 2004). There is an urgent demand for other sources than the primary source plant, whose wild populations are being destroyed more and more and the pharmaceutical demand will not be covered anymore if no other sources are made available (Eyberger et al., 2006).

OH O O O N O N O O

H3CO OCH3 HO O OCH3 camptothecin podophyllotoxin

1.4. Compounds from Terrestrial Fungi in Clinical Trials

There are currently two fungal-derived molecules that serve as templates for analogs used in clinical trials, fumagillin and illudin S (Buttler, 2005). Fumagillin arises from a mixed sesquiterpenoid (C15-nucleus) and polyketidic (C10 side chain) biosynthesis and is produced by the fungus Aspergillus fumigatus (Birch & Hussain, 1969). It has been the template for analogues undergoing oncological clinical trials. Fumagillin is an angiogenesis inhibitor, which suppresses the formation and growth of new blood vessels, namely the angiogenesis, by direct blocking endothelial cell proliferation (Ingber et al., 1990). Semi-synthetic derivatives, such as TNP-470, PPI-2458 and CKD- 732 entered clinical trials for the treatment of breast, prostate, and brain cancer, as well as Kaposi sarcoma (Kruger & Figg, 2000, Lee et al., 2004; Kim et al., 2007, Bernier et al., 2005; Chun et al., 2005; Ingber et al., 1990). TNP-470 acts through an irreversible binding to methionine aminopeptidase 2, which then leads to intracellular signalling interference (Zhang et al., 2006; Lu et al., 2006). Although clinical trials of the fumagillin derivative PPI-2458 against cancer were later terminated (Buttler, 2008), the

same antiproliferative mechanism of action might be used for the treatment of rheumatoid arthritis, and PPI-2458 has been investigated towards this disease. Furthermore, fumagillin has been proven to be highly active as antimicrosporidial agent in mice (Didier et al., 2006) and has been found to inhibit HIV-1 viral protein R (VPR) activity (Asami et al., 2006). Preliminary evidence has recently been put forward that angiogenesis inhibitors also inhibit the growth of atherosclerotic plaques (Moulton, 1999). Remarkably, fumagillin was approved for use in the treatment of intestinal microsporidiosis in France in 2005, a disease caused by the parasite Enterozytozoon bieneusi, which is of major concern to immunocompromised patients as it can cause diarrhoe (Burton, 2002; Alvarado et al., 2009).

O H

O OMe O COOH

O fumagillin

O H O CH3 O H H O O O OMe O O OMe N OMe O N NH O O N H 2 H Cl O O O O PPI-2458 CKD-732 TNP-470

Another anticancer lead compound of fungal origin is the sesquiterpene illudin S, which was first isolated from the basidiomycete Omphalotus illudens (formerly Clitocybe illudens) and was shown to be extremely cytotoxic against the human leukemia cell line

HL-60 in vitro (IC50 = 3 ng/mL), but exhibiting a low therapeutic index in mouse xenograft solid tumor systems (Anchel et al., 1950; McMorris & Anchel, 1965; McMorris et al., 1996). Though, the improved semi-synthetic compound irofulven has a significantly superior therapeutic index in comparison to the parent natural product illudin S and possesses selectivity towards human tumor cells in which apoptosis is induced (e.g. MV522 lung carcinoma cell assay with an IC50 value of 73 nM for irofulvene), with only a marginal apoptotic effect on normal human cells (Baekelandt, 2002; Woynarowska et al., 2000, McMorris 2001). Irofulvene possesses a greater

efficacy than illudin S due to its slower and more selective action. It has entered phase I and II clinical trials against several types of cancer (Seiden et al., 2006).

OH OH

HO OH HO O O

iIlludin S irofulven

1.5. Marine Natural Products as Pharmaceutical Drugs

The marine ecosystem, covering more than 70 % of the planet’s surface, is home to an immense diversity of life. Though, due to the extreme depth of the oceans, they represent more than 99 % of our planet’s natural habitat when setting the terrestrial habitat up to 100 m of height (Schuh, 2008). Marine natural products are conspicuously absent in table 1, as their systematic exploration became widespread only recently. In the United States there are three FDA approved marine-derived drugs, namely cytarabine, vidarabine and ziconotide. Currently, trabectedin has been approved by the European Agency for the Evaluation of Medicinal Products (EMEA), and is completing key Phase III studies in the US for approval. The current clinical pipeline includes 13 marine-derived compounds that are either in Phase I, Phase II or Phase III clinical trials (Mayer et al., 2010).

Table 2, Marine-derived compounds in clinical development and approved as pharmaceutical drugs (Mayer et al., 2010) Clinical status Compound name Structural class Origin Disease area Approved Cytarabine, Ara-C Nucleoside Sponge Cancer Vidarabine, Ara-A nucleoside Sponge Antiviral Ziconotide Peptide Snail Pain Trabectedin (ET-743) Alkaloid Tunicate Cancer (EU registered only) Phase III Eribulin Mesylate (E7389) Macrolide Sponge Cancer Soblidotin (TZT 1027) peptide Bacterium Cancer Phase II DMXBA (GTS-21) Alkaloid Worm Cognition/Schizophrenia Plinabulin (NPI-2358) Diketopiperazine Fungus Cancer Plitidepsin Depsipeptide Tunicate Cancer Elisidepsin Depsipeptide Mollusc Cancer PM1004 Alkaloid Nudibranch Cancer Tasidotin, Synthadotin (ILX-651) Peptide Bacterium Cancer Pseudopterosins Diterpene glycoside Soft coral Wound healing Phase I Bryostatin 1 Polyketide Bryozoa Cancer Hemiasterlin (E7974) Tripeptide Sponge Cancer Marizomib (Salinosporamide A; Beta-lactone- NPI-0052) gamma lactam Bacterium Cancer

A recent development in the area of antitumor agents from natural sources was the approval of the alkaloid trabectedin for advanced soft HO

tissue sarcoma (Verweij, 2009) and patients with NH H3CO OCH relapsed platinum-sensitive ovarian cancer (Yap et O 3 O HO al., 2009) by the EMEA (European Medicines O S H O H Agency) in September 2007 N N (http://www.emea.europa.eu). Currently the product O H O OH is being developed in Phase II trials in breast, lung, trabectedin (ET-743, Yondelis) prostate and pediatric cancer, and Phase III trials for first-line therapy in STS (Mayer et al., 2010). Yondelis® is being developed and marketed by Pharmamar (http://www.pharmamar.com/products.aspx). Trabectedin, was originally isolated from a marine invertebrate, the tunicate Ecteinascidia turbinata which was found in the Caribbean and Mediterranean sea (Rinehart, et al. 1990, Wright, et al. 1990), but is now produced semi-synthetically (Cuevas et al., 2000; Newman et al., 2000; Rinehart et al., 1990; Wright et al., 1990; http://www.pharmamar.com). Trabectedin binds guanine-specific to the minor groove of DNA, but its precise mechanism of action remains poorly understood (Herrero et al., 2006; Pommier et al., 1996).

Cytarabine (Ara-C, developed by Bedford Laboratories, http://www.bedfordlabs.com/, and Enzon Pharmaceuticals, http:// www.enzon.com/), is a synthetic pyrimidine nucleoside which was developed from spongothymidine, a NH2 nucleoside originally isolated from the Caribbean sponge Tethya HN crypta (Newman et al., 2009). Cytarabine is an S-phase specific O N antimetabolite cytotoxic agent, which when converted HOH2C O intracellularly to cytosine arabinoside triphosphate competes with HO the physiologic substrate deoxycitidine triphosphate, thus resulting OH in both inhibition of DNA polymerase and DNA synthesis. Cytarabine (Ara-C) Cytarabine received FDA approval in 1969. FDA-labeled indications for conventional cytarabine are treatment of acute lymphocytic leukemia, acute myelocytic leukemia, blast crisis phase of chronic myelogenous leukemia, meningeal leukemia (Thomas, 2009; Absalon et al., 2009; MARTINDALE, 2009) and intrathecal treatment of lymphomatous meningitis. Vidarabine (Ara-A) is a synthetic purine nucleoside which was developed from spongouridine, a nucleoside originally isolated from the Caribbean sponge Tethya crypta (Newman, et al., 2009), and which is currently obtained NH2 from Streptomyces antibioticus. Adenine arabinoside inhibits viral N N

DNA polymerase and DNA synthesis of herpes, vaccinia and N N HOH2C varicella zoster viruses. Although its marketing status is currently O HO listed as discontinued by the FDA in the US market, vidarabine (Vira-A1) received FDA approval in 1976. FDA-labeled OH indications for conventional vidarabine (Vira-A ophthalmic Vidarabine (Ara-A) ointment, 3%) are treatment of acute keratoconjunctivitis, recurrent epithelial keratitis caused by herpes simplex virus type 1 and 2, and superficial keratitis caused by herpes simplex virus that has not responded to topical idoxuridine (Herplex1) (MARTINDALE, 2009). Vidarabine (Vira-A1), previously marketed by King Pharmaceuticals (http://www.kingpharm.com/) was discontinued in June of 2001 by an executive decision, possibly associated with the lower therapeutic window of vidarabine relative to newer antiviral compounds currently on the market (Mayer et al., 2010).

Ziconotide (Prialt®, Elan Corporation, PLC (http://www.elan.com/therapies/products/prialt.asp) is the synthetic equivalent of a naturally occurring 25-amino acid peptide, ω-conotoxin MVIIA, originally isolated from the venom of the fish-hunting marine snail Conus magus (Olivera, 2000). Receiving FDA-approval in 2004, Ziconotide is a potent analgesic with a completely novel mechanism of action (Bingham et al., 2010; Kerr and Yoshikami, 1984). Various subtypes of voltage-gated calcium channels have been identified in the nervous system. Ziconotide reversibly blocks N-type calcium channels located on primary nociceptive afferent nerves in the superficial layers of the dorsal horn of the spinal cord. Binding of ziconotide to presynaptic N-type calcium channels reduces the release of excitatory neurotransmitter release from the primary afferent nerve terminals (Miljanich, 2004; McGivern, 2006). Tolerance to drug effects is a major limiting factor in opiate-based therapies; unlike opiates, ziconotide does not produce tolerance (Wang et al., 2000; Gaur et al., 1994). Ziconotide is currently used for the management of severe chronic pain in patients with cancer or AIDS (Staats et al., 2004; Rauck et al., 2009), for whom intrathecal (IT) therapy is warranted, and who are intolerant or refractory to other treatments, such as systemic analgesics, adjunctive therapies or IT morphine.

Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2

ω-Conotoxin MVIIA

1.6. Marine-derived fungal compounds in clinical trials

As interests have turned to marine microorganisms, marine fungi have proved to be a rich and promising source of novel bioactive natural products. Most of these microorganisms grow in a unique and extreme habitat and therefore have the capability to produce unusual secondary metabolites. It is believed that the metabolites possibly act as a chemical defense adaptation of fungi competing for substrates. The production of these unique secondary metabolites by marine fungi is most likely due to their adaptation to a very distinct set of environmental pressures (Bhadury et al., 2006).

Although some marine-fungal compounds are in pre-clinical trials, the most promising marine-fungal derived analogue in clinical trials is the anticancer agent plinabulin which is based on the diketopiperazine halimide (also known as phenylahistidin), isolated from the fungus Aspergillus ustus (Kanoh et al., 1997). Initially halimide was isolated as a mixture of enantiomers, but the (-) enantiomer alone exhibited a 50-fold increase of in vitro antitumor activity against human cancer cell lines, along with inhibition of tubulin polymerisation by interacting with the colchicine-binding site on tubulin (Kanoh et al., 1999a & 1999b).

O O

NH N NH N HN NH HN NH

O O

(-)-halimide plinabulin To optimise biological activity, a series of 200 synthetic analogues were generated by the company Nereus Pharmaceuticals, San Diego, U.S.A., and finally led to compound NPI-2358 (Plinabulin), which is since 2009 in Phase II clinical studies as a vascular disrupting agent against advanced non-small cell lung cancer refractory to current therapy (http://www.nereuspharm.com). A recent study showed that plinabulin acts moreover as a tumor vascular-disrupting agent by inducing rapid depolymerisation of existing microtubules in the highly proliferating tumor vascular endothelial cells, as evidenced by in vitro assays with human umbilical vein endothelial cells (HUVECs) (Nicholson et al., 2006). Tumor vascular-disrupting agents thereby attack established tumor blood vessels, resulting in occlusion of vasculature in the tumor, which induces tumor cell hypoxia and finally necrosis.

2. Scope of the present study

Due to the rapidly increasing number of pathogenic bacteria, viruses and tumor cells that possess resistance towards established therapies, lead structures for the development of new drugs are in high demand. Marine microorganisms, such as fungi often occur associated with macroorganisms like algae, sponges or tunicates and produce secondary metabolites with novel structures and potential pharmaceutical significance.

The goal of this study was the evaluation of new natural products of marine-derived fungi in order to find novel lead structures for drug development, with a focus on anticancer leads. To achieve this aim seven fungal strains living associated with marine algae and sponges were cultivated during 40 to 60 days, and the extracts tested for bioactivity. Subsequently, the natural products were isolated, their structures determined and their bioactivity established.

The research relies in tracing active principles from the selected extracts by performing NMR guided fractionation, together with the application of various preparative chromatographic systems (e.g., VLC, HPLC). The structure elucidation of the obtained pure compounds relies on interpretation of 1D and 2D NMR spectroscopic data, as well of mass spectrometry data, UV, IR and CD spectra. Additionally, the isolated compounds can be submitted to a vast panel of biological assays, such as antimicrobial, antiplasmodial, antidiabetic, antiinflammatory, antiviral or psychoactive activity, amongst others.

3. General Methodology

3.1. General Experimental Procedures

Optical rotations were measured on a Jasco DIP 140 polarimeter. UV and IR spectra were obtained employing a Perkin-Elmer Spectrum BX instrument. CD spectra were recorded in MeOH at room temperature using a JASCO J-810-150S. All NMR spectra were recorded in MeOD, CDCl3 or (CD3)2CO employing a Bruker Avance 300 DPX spectrometer. Spectra were referenced to residual solvent signals with resonances at δH/C

3.35/49.0 for MeOD, δH/C 7.26/77.0 for CDCl3 and δH/C 2.04/29.8 for (CD3)2CO. HREIMS were recorded on a Finnigan MAT 95 spectrometer. HRESIMS were recorded on a Bruker Daltonik micrOTOF-Q Time-of-Flight mass spectrometer with ESI source. HPLC was carried out using a system composed of a Waters 515 pump together with a Knauer K-2300 differential refractometer. HPLC columns were from Knauer (250 x 8 mm, 5 μm, Eurospher-100 Si and 250 x 8 mm Eurospher-100, C18, 5 μm, flow rate 2 mL/min) and from Macherey-Nagel (Nucleodur C18 EC Isis and Nucleodur C18 Sphinx RP, both with 250 x 4.6 mm, 5 μm, flow rate 1 mL/min). Merck silica gel 60 (0.040–0.063 mm, 70-230 mesh) was used for vacuum liquid chromatography (VLC). Columns were wet-packed under vacuum using petroleum ether (PE). Before applying the sample solution, the columns were equilibrated with the first designated eluent. Standard columns for crude extract fractionation had dimensions of 13 x 4 cm.

3.2. Fungal material:

Marine-derived fungus Stachylidium sp. was isolated from the sponge Callyspongia sp. cf. C. flammea and identified by Dr. P. Massart and Dr. C. Decock from the Belgian coordinated collections of microorganisms of the Catholic University of Louvain, (BCCM/MUCL). A specimen is deposited at the Institute for Pharmaceutical Biology, University of Bonn, isolation number “293K04”, running number “220”.

The marine-derived fungus Cadophora malorum (Kidd & Beaumont) W. Gams was isolated from the green alga Enteromorpha sp. and identified by P. Massart and C. Decock, BCCM/MUCL, Catholic University of Louvain, Belgium. A specimen is deposited at the Institute for Pharmaceutical Biology, University of Bonn, isolation number “SY3-1-1MIT”, running number “417”.

The marine-derived fungus Verticillium tenerum was isolated from an unknown alga and identified by Isabelle Charue, BCCM/MUCL, Catholic University of Louvain, Belgium. A specimen is deposited at the Institute for Pharmaceutical Biology, University of Bonn, isolation number “293K04”, running number “780”.

3.3. Methodology for the performed bioactivity assays

The referred compounds were tested in antibacterial (Escherichia coli, Bacillus megaterium), antifungal (Mycotypha microspora, Eurotium rubrum, and Microbotryum violaceum), and antialgal (Chlorella fusca) assays as described by Schulz et al., 1995 and Schulz et al., 2002. Compounds were tested towards a panel of proteases including chymotrypsin, trypsin, human leukocyte elastase (HLE), papain, porcine cease, and acetylcholine esterase as described by Neumann, et al., 2009. Compounds were tested for protein kinases inhibition assays (DYRK1A and CDK5) according to Bettayeb et al., 2008. The triglyceride accumulation inhibition in the 3T3-L1 murine adipocytes assay were performed as described by Shimokawa et al., 2008. Cytotoxic activity assay against a panel of 5 cancer cell lines, NCI-H460/lung, A549/lung, MCF7/breast and SF268/CNS and CAKI/renal at the 100 µM level was performed according to Saroglou et al., 2005 and Monks et al., 1991. Compounds were tested for antiplasmodial activity assay against Plasmodium berghei as described by Prudêncio et al., 2008. The sporogenic activity assay was performed according to Kitahara et al., 1984 and Sawai et al., 1985.

Inhibition of the viral HIV-1 and HIV-2 induced cytopathogenic effect in MT-4 cells assays were performed according to Pannecouque et al., 2008 and Zhan et al., 2008. Severe Acute Respiratory Syndrome coronavirus (SARS) assays were performed according to Kumaki et al., 2008. The Herpes Simplex Virus-2 (HSV-2) activity assay methods are described by Harden et al., 2009. The Respiratory Syncytial virus (RSV) activity assays were conducted according to Barnard et al., 1993 and Barnard et al., 1997. Two influenza viruses (Flu A and Flu B) activity assays were performed as described by Sidwell and Smee, 2000. Hepatitis B virus assay was performed according to Sells et al., 1998 and Korba and Gerin, 1992. The Epstein-Barr virus (EBV) assay was performed according to Smee et al., 1997. The activity assays against two strains of antibiotic resistant Mycobacterium tuberculosis were performed according to Bauer et al., 1966. The methodology for the inhibition of the NF-kB protein complex is described by Schumacher et al., 2010. The compounds were tested against a panel of anti-diabetic activity assays as described by Marrapodi and Chiang, 2000, Dey et al., 2007 and Seale et al., 1997. The agonistic/antagonistic binding assays against a panel of forty four psychoactive receptors (activity considered with at least 50 % inhibition at the 10 μM level against 5HT1A, 5HT1B, 5HT1D, 5HT1E, 5HT2A, 5HT2B, 5HT2C, 5HT3, 5HT5A, 5HT6, 5HT7, Alpha1A, Alpha1B, Alpha1D, Alpha2A, Alpha2B, Alpha2C, Beta1, Beta2, Beta3, BZP Rat Brain Site, D1, D2, D3, D4, D5, DAT, DOR, Gaba A, H1, H2, H3, H4, KOR, M1, M2, M3, M4, M5, MOR, NET, SERT, Sigma1, Sigma2). Ocasionally the compounds were also tested for specific receptors, i.e., CB1, CB2, GabaB, NMDA-PCP site, Oxytocin, PBR, mGluR5_Rat Brain, V1A, V1B and V2. All experimental procedures are desribed by the assay provider at http://pdsp.med.unc.edu/UNC- CH%20Protocol%20Book.pdf.

4. Marilines A – D, Novel Phthalimidines from the Sponge-derived Fungus Stachylidium sp.

Abstract The marine-derived fungus Stachylidium sp. was isolated from the sponge Callyspongia cf. C. flammea. Chemical investigation of the bioactive fungal extract led to the isolation of the novel phthalimidine derivatives marilines A - D (1-4). The absolute configuration of the enantiomeric compounds 1 and 2 was assigned by means of theoretical Circular Dichroism calculations. The skeleton of marilines A - D is most unusual, and its biosynthesis is suggested to require unusual biochemical reactions in fungal secondary metabolism. Both enantiomers marilines A and B (1, 2) inhibited human leucocyte elastase (HLE) with an IC50 value of 0.86 μM, and acetylcholinesterase (AChE) with IC50 values of 0.18 μM (offset = 49 %) and 0.63 μM (offset = 54 %), respectively.

9 R2 17 R1 6 6 O 3' 5' 8' O 8' 12 8 12 O N 1' N 1' 1 9' 1 18 14 3 11 10' HO 10' O 13 O O O O 1'' 10 4'' (3) (1) R =CH ;R =H 5'' 1 3 2

(2) R1 =H; R2 =CH3 OH HN

O O (4)

Figure 1. Structural Formulae of Compounds 1 – 4

Introduction

Inspired by the impressive level of biodiversity in the marine environment, the pharmacological potential of natural products from marine organisms has been investigated enthusiastically (Glaser and Mayer, 2009). Phthalimidines are secondary metabolites mostly isolated from fungi, but also reported from plants and bacteria. Fungal-derived phthalimidines are described as having a wide spectrum of bioactivities, e.g. the phytotoxic activity of cichorine, a metabolite isolated from Alternaria cichorii and Aspergillus silvaticus that causes necrotic lesions in Russian knapweed (Kawahara et al., 1988; Stierle et al., 1993). Porritoxin is known to be a non-specific toxin isolated from Alternaria porri, inhibiting the growth of lettuce and stone-leek roots, two important commercial plants (Suemitsu et al., 1992; Suemitsu et al., 1995; Horiuchi et al., 2003; Horiuchi et al., 2002; Moreau et al., 2006). Stachybocins and spirodihydrobenzofuranlactams were isolated from Stachybotrys sp. and are reported to possess endothelin receptor antagonistic effects. The latter has also HIV-1 protease inhibitory properties (Ogawa et al., 1995; Roggo et al., 1996). The antifungal pestalachlorides isolated from Pestalotopsis adusta (Li et al., 2008), the aldose reductase inhibitors salfredins from Crucibulum sp. (Matsumoto et al., 1995) or the cytotoxic hericenone B isolated from Hericium erinceum (Kawagishi et al., 1990) additionally demonstrate the broad spectrum of bioactivity found for fungal-derived phthalimidine-like structures.

During our search for new natural products produced from the marine-derived fungus Stachylidium sp., four novel pthalimidine derivatives, marilines A-D, were isolated from a culture grown on agar-biomalt medium supplemented with artificial sea salt. Albeit phthalimidine-like structures are not rare, the structural skeleton of marilines A - D is most unusual, and its biosynthesis is suggested to require unique reactions in fungal secondary metabolism. Marilines A-D were tested in a broad range of assays to determine their biological activity. Both enantiomers, marilines A and B (1, 2) inhibited human leucocyte elastase (HLE) with the same potency (IC50 0.86 μM).

Acetylcholinesterase (AChE) was inhibited with an IC50 of 0.18 μM (offset = 49 %) and 0.63 μM (offset = 54 %), for marilines A and B, respectively. Compounds 1 and 2 also showed weak antiplasmodial and antiproliferative activity, and were antagonistic in several assays employing psychoactive receptors.

Results

Compounds 1 and 2 were initially obtained and analysed as a mixture of enantiomers. The molecular formula was deduced by accurate mass measurement (HREIMS) to be 13 C33H43NO5, requiring thirteen degrees of unsaturation. The C NMR and DEPT135 spectra contained 33 carbon resonances, including nine resulting from methyl groups, seven from sp2 methines, one from a sp3 methine, further four signals from methylene groups and 12 resonances were assigned to quaternary carbons. 1H NMR and 1H-1H COSY spectra showed four resonance signals in the aromatic region (δ 6.95 s, 6.67 d, 6.56 dd, 7.19 d) indicating together with 13C NMR and 1H-13C HMBC data the presence of two benzylic moieties, one of them 1,2,4- and the other penta-substituted (Tables 1, 2 and Appendix - 2D NMR Tables). Furthermore, the molecule was found to contain three 1,1,2-substituted double bonds (δC 120.6, 141.5, 124.6, 132.0, 120.8, 137.9), which were part of a mono- and a hemiterpene moiety. The C-1’ to C-10’ part of the molecule was deduced from two proton coupling spin systems, the first from H-2’ to 1 13 H2-1’ and H3-9’, and the second ranged from H-4’ through to H3-10’ and H3-8’. H- C

HMBC data correlated H3-9’ to C-2’, C-3’ and C-4’, which disclosed the structure of this terpenoid fragment. Based on literature comparisons (Tanaka et al., 1985) we established the configuration of the 2’/3’ as E. 1H NMR resonance signals that arose from the hemiterpene unit C-1’’ to C-5’’ included two singlet methyl resonances, i.e. 1 13 CH3-4’’(δH 1.68, brs) and CH3-5’’(δH 1.67, brs), both with H- C HMBC correlations 2 to C-3’’ and the sp methine CH-2’’ (δH 5.35). H-2’’ coupled to the oxygenated methylene protons H2-1’’, thus completing this partial structure. A 13C NMR signal at δ 57.5 (CH-8) was found to be characteristic for a carbon neighboring a nitrogen atom, which according to its chemical shift in the 15N NMR 1 1 spectrum (δN 132.4) was part of an amide functionality. The H- H COSY showed 1 13 correlations from H-8 to H3-9. Furthermore, H- C HMBC spectra showed correlations between H-8 and the carbonyl carbon C-1, as well as to C-6, C-7 and C-2 of the penta- 1 13 substituted aromatic ring. H- C HMBC correlations between H3-9 and C-7 as well as 1 15 H- N HMBC correlations between H3-9 and the amide nitrogen (see Appendix - Complete 2D NMR data) gave evidence for a phthalimidine skeleton, i.e. the C-1 to C-9 part of the structure. One of the substituents of the aromatic ring of the phthalimidine moiety was established to be a methyl group (CH3-11, δH 2.12, s), which was positioned at C-4 as deduced from HMBC correlations (Tables 1 and 2). The quaternary carbons

C-3 and C-5 have 13C NMR shifts typical for aromatic carbons bound to oxygen. The 1 13 methoxyl group OCH3-10 was established to be located at C-3, as evident from H- C

HMBC correlations. CH2-1’ of the monoterpene moiety was attached to oxygen as concluded from the chemical shifts of C-1’ (δC 66.2) and H2-1’ (δH 4.68 and 4.72). H2- 1’ showed long range heteronuclear coupling with C-5 of the aromatic ring via an oxygen bridge, thus placing the terpene moiety at C-5. The latter deduction was also 1 1 supported by H- H NOESY correlations from H2-1’ to H-6. Further three resonance signals in the 1H NMR spectrum resulted from a tri-substituted benzene ring. H-16 and H-17 (J = 8.6 Hz) were placed in ortho position to each other, while H-14 and H-16 (J = 2.6 Hz) were meta-positioned. 1H-13C HMBC correlations from H2-1’’ to C-13 and from OCH3-18 to C-15 showed that the oxygenated quaternary aromatic carbons C-13 (δC 156.8) and C-15 (δC 160.8) were connected with a 1 1 hemiterpene (C-1’’ to C-5’’) and a methoxyl moiety (OCH3-18), respectively. H- H

NOESY correlations of H3-18 to H-14 and H-16, and of H2-1’’ to H-14 confirmed the position of these substituents. 1H-15N HMBC measurements showed two distinct correlations from H3-9 and H-17 to the nitrogen atom (δN 132.4), evidencing that the tri- substituted benzene ring is connected at C-12 to the phthalimidine nucleus. 1H-1H

NOESY correlations from H-17 to H3-9 and to H-8 confirmed the structure. Compounds 1 and 2 have the same planar structure, but differ concerning their configuration at C-8, i.e. they are enantiomers. We propose the trivial names mariline A (1) and B (2) for these compounds.

The molecular formula of 3 was deduced by accurate mass measurement (HRESIMS) to be C23H33NO4, requiring eight degrees of unsaturation. The C-1 to C-8 and C-1’ to C- 10’ parts of the molecule were established to be identical to those of compounds 1 and 2 based on 13C and 1H NMR, as well as 1H-13C HMBC and 1H-1H COSY spectra (Tables 1, 2 and Appendix - 2D NMR tables) The structure of 3 however, was missing the second aromatic ring and instead an ethyl-1-ol moiety was linked to the nitrogen atom.

This was evidenced by heteronuclear long range correlations from H2-12 to C-8 and C-1 through the nitrogen atom. Furthermore, the methylene CH2-13 showed proton coupling 13 with CH2-12, whereby the C NMR shift of C-13 indicated that it was connected to a hydroxy group (δC 61.7). This completed the planar structure of compound 3, for which we propose the trivial name mariline C.

The molecular formula of 4 was deduced by accurate mass measurement (HRESIMS) to be C11H13NO3, requiring six degrees of unsaturation. NMR data (Tables 1, 2 and Appendix - 2D NMR tables) suggested the presence of a phthalimidine nucleus with most substituents on the aromatic ring as present in 1, 2 and 3. Compound 4, however possessed a free hydroxy group, which was confirmed by an IR absorption band at 3346 -1 cm . The latter was placed at C-5 (δC 162.3), which was clearly an oxygenated aromatic carbon atom. Spectroscopic data for compound 4 did not show any signals for a substituent on the nitrogen atom, which is thus present as an NH group. We propose the trivial name mariline D for 4.

Table 1. 13C NMR Spectroscopic Data for Compounds 1 – 4 1 & 2 3 4 a, b, c a, b, c a, b,c position δN, δC, mult. δC, mult. δC, mult. N 132.4, N 1 166.2, qC 167.3, qC 171.7, qC 2 116.4, qC 116.3, qC 114.5, qC 3 157.2, qC 156.9, qC 158.4, qC 4 119.4, qC 119.3, qC 118.9, qC 5 161.9, qC 161.7, qC 162.3, qC 6 101.8, CH 101.8, CH 105.0, CH 7 150.0, qC 150.0, qC 152.1, qC 8 57.5, CH 56.9, CH 53.2, CH

9 19.4, CH3 18.9, CH3 20.8, CH3 10 62.1, CH3 62.1, CH3 62.5, CH3 11 8.8, CH3 8.8, CH3 8.6, CH3 12 120.1, qC 43.6, CH2 13 156.8, qC 61.7, CH2 14 101.2, CH 15 160.8, qC 16 105.5, CH 17 131.9, CH

18 55.7, CH3 1' 66.2, CH2 66.2, CH2 2' 120.6, CH 120.6, CH 3' 141.5, qC 141.5, qC

4' 40.1, CH2 40.1, CH2 5' 27.0, CH2 27.0, CH2 6' 124.6, CH 124.6, CH 7' 132.0, qC 132.1, qC

8' 25.8, CH3 25.8, CH3 9' 16.7, CH3 16.7, CH3 10' 17.7, CH3 17.7, CH3 1'' 66.1, CH2 2'' 120.8, CH 3'' 137.9, qC

4'' 18.2, CH3 5'' 25.7, CH3 a b acetone-d6 (1, 2, 3) , and MeOD (4) 300/75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments (1H-13C, 1H-15N HMBC, HSQC, COSY). c Implied multiplicities determined by DEPT.

Table 2. 1H NMR Spectroscopic Data for Compounds 1 – 4 1 & 2 3 4 a, b a, b a, b position δH (J in Hz) δH (J in Hz) δH (J in Hz) N 1 2 3 4 5 6 6.95, s 6.92, s 6.68, s 7 8 4.97, q (6.8) 4.62, q (6.8) 4.53, q (6.8) 9 1.27, d (6.8) 1.44, d (6.8) 1.42, d (6.8) 10 3.99, s 3.98, s 3.95, s 11 2.12, s 2.09, s 2.15, s 12 a: 3.33, dt (14.0, 5.5) b: 3.85, dt (14.0, 5.5) 13 3.71, m 14 6.67, d (2.6) 15 16 6.56, dd (2.6, 8.6) 17 7.19, d (8.6) 18 3.81, brs 1' a: 4.68, dd (6.6, 12.3) a: 4.65, dd (6.6, 12.3) b: 4.72, dd (6.6, 12.3) b: 4.71, dd (6.6, 12.3) 2' 5.53, t (6.6) 5.51, t (6.6) 3' 4' 2.12, m 2.12, m 5' 2.14, m 2.14, m 6' 5.10, t (6.6) 5.10, t (6.6) 7' 8' 1.63, brs 1.63, brs 9' 1.77, brs 1.76, brs 10' 1.59, brs 1.58, brs 1'' a: 4.54, dd (6.6, 12.0) b: 4.58, dd (6.6, 12.0) 2'' 5.35, t (6.6) 3'' 4'' 1.68, brs 5'' 1.67, brs a b acetone-d6 (1, 2, 3) , and MeOD (4), 300/75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments (1H-13C, 1H-15N HMBC, HSQC, COSY).

Stereochemistry

Compounds 1-4 possess a single chiral carbon, i.e. C-8. The measurement of specific optical rotations yielded in all cases a value close to zero, and furthermore, CD measurements produced only marginal CD effects. The structure of the molecules suggested a CD effect at around 260 nm due to the proximity of the chiral center to the chromophore. It was thus assumed that we may have obtained racemic mixtures. In the case of marilines A and B (1, 2) this was confirmed by chromatographic separation of the enantiomers using chiral HPLC. The enantiomers 1 and 2 were isolated from three independent cultures and found to be present in ratios of approximately 3:2, 3:7 and 1:1 as deduced from the final weights obtained.

We found several previously reported cases of racemization reactions on natural products with a chiral center in lactam, lactone and dihydrofuran rings (Harper et al., 2003; Li et al., 2008; Strobel, et al., 2002; Wolfgang and Kund, 1990), which suggested a post-biosynthetic racemization also for 1 or 2. This racemization could occur via a resonance stabilized cationic intermediate, as previously described for similar compounds (Harper et al., 2003; Li et al., 2008). Especially noteworthy is a previous literature report on the easy racemization of the phthalimidine-like compound pagoclone (Stuck et al., 2003). It was reported that this isoindolinone already racemized at low basic or acidic conditions and moderately elevated temperatures. Racemization of pagoclone was found to be optimal with 0.005N KOH at 50 ºC for 2h, and suggested to occur via a retro-Michael/Michael reaction. This type of reaction is, however unlikely to occur in the case of 1 and 2. Nevertehless, to evaluate whether racemization occured during the extraction and isolation process, enantiomerically pure marilines A and B were subjected to increasing concentrations of KOH and HCl as outlined in the experimental section, however even harsh conditions produced no racemization as evidenced by chiral HPLC analysis.

CD spectra of 1 and 2 were measured and opposite CD effects were discerned for the enantiomers (Figure 2).

(Compound 1)

(Compound 2)

Figure 2. CD spectra of compound 1 (mariline A) and 2 (mariline B)

The configuration at C-8 is currently being elucidated by theoretical CD calculations.

Compounds 3 and 4 were also collected as racemic mixtures, as no effects in CD spectra and no optical rotation values were obtained. Exhaustive attempts to separate the enantiomers with three different chiral stationary HPLC columns were unsuccessful.

Biological activity

Marilines A and B inhibited the protease HLE with an IC50 value of 0.86 μM for both enantiomers. Acetylcholinesterase (AChE) activity was inhibited with an IC50 value of 0.18 μM (offset = 49 ± 3 %) for mariline A and 0.63 μM (offset = 54 ± 3 %) for mariline B. The large offset (i.e. a remaining activity in the presence of inhibitor) in the AChE inhibition assay probably means that the compounds do not bind to the protease active site. When performing calculations with equations which do not considers the offset, marilines A and B have IC50 values of 4.4 and 6.6 μM, respectively. Mariline C (3) showed no activity toward the proteases HLE and AChE at the 10 μM level (see Appendix - Bioactivity results). Compound 4 was not tested in these assays, due to the small amounts isolated. Marilines A-D were assayed towards further proteases and found to be inactive (see Appendix - Bioactivity results).

Mariline A was tested against five cancer cell lines and exhibited a mean GI50 of 24.44

μM, whilst mariline B was tested against 19 cancer cell lines and showed a GI50 of 5.00 μM. The observed antiproliferative pattern of mariline B did not correlate with that of any of the standard antiproliferative compounds with known mechanism of action as deduced by COMPARE analyses. The first match on the standard agents database (correlation 0.427) was rhizoxin, a metabolite produced by a bacteria associated with a fungus (see see Appendix - Bioactivity results). Mariline C (3) and D (4) were also evaluated for cytotoxic activity against a panel of 5 cancer cell lines (NCI-H460/lung, A549/lung, MCF7/breast and SF268/CNS and CAKI/renal at the 100 µM level) and mariline C exhibited low antiproliferative activity with an GI50 of 48.32 μM, while mariline D exhibited no activity.

Marilines A-C were shown to have antiplasmodial activity against Plasmodium berghei with an IC50 of 6.68 μM, 11.61 μM and 13.84 μM, respectively.

Marilines C and D showed also antagonist activity on the canabinoid receptor CB2 with

Ki values of 5.97 μM and 5.94 μM, respectively. Mariline B further exhibited antagonist activity against the histamine receptor H2 with a

Ki value of 5.92 μM, the dopamine receptor DAT with a Ki value of 5.63 μM and the adrenergic receptor Beta3 with a Ki value of 5.63 μM (see Appendix - Bioactivity results).

Mariline A and B were further evaluated for inhibition of the protein kinases DYRK1A and CDK5 assays (tested at 10 mM dose) and for the inhibition of further proteases: Chymotrypsin, trypsin, papain and acetylcholine esterase (tested at the 100 µM level), but showed no activity. Mariline B (2) was tested against two antibiotic resistant Mycobacterium tuberculosis, strains MTB72 and R46 (tested at the 50 µg/mL disc level), but did not show relevant activity.

Discussion

Compounds with a related phthalimidine (isoindolinone) nucleus as found for 1 – 4 are known from fungal metabolism, however they all lack the methyl group at C-8. The structurally most simple phthalimidines possess an unsubstituted nitrogen atom as found in mariline D (4), i.e. 6-hydroxy-4-methoxy-5-methylphthalimidine (cichorine) (Kawahara et al., 1988; Stierle et al., 1993), zinnimidine (Suemitsu et al, 1995), duricaulic acid (Achenbach et al., 1985), memnobotrin A and the related staybotrylactam (Jarvis et al., 1999) and the more complex xylactam (Wang et al, 2005). Reported N-substituted phthalimidines include compounds with a phenylethyl (Kawagishi et al., 1990; Nozawa et al., 1997), ethyl-1-ol (Horiuchi et al., 2003; Horiuchi et al., 2002) or a carbonic acid (Ogawa et al, 1995, Matsumoto et al., 1995) moiety attached. Of these, examples of fungal phthalimidines structurally related to marilines A and B (1, 2) include hericenone B and stachybotrin C (Kawagishi et al., 1990; Nozawa et al., 1997), both with a phenylethyl moiety connected to the nitrogen atom. In compounds 1 and 2, however the nitrogen is substituted with a derivatised phenyl ring, hitherto only present in a similar way in the phthalimidine structure clitocybin A (Kim et al, 2008). Porritoxin, porritoxin sulfonic acid (Horiuchi et al., 2003; Horiuchi et al., 2002), memnobotrin B (Jarvis et al., 1999) and stachybotramide (Ayer et al., 1993) contain an N-ethyl-1-ol moiety and are structurally related to mariline C (3) (all related structures are depicted in see Appendix – Spectroscopic data supporting information)

Racemization

8R and 8S enantiomers of phthalimidines are described for pestalachloride A, where a 2,4-dichloro-5-methoxy-3-methylphenol moiety is attached to the chiral carbon C-8 (Li et al., 2008). Also, the isobenzofuranone isopestacin was found to occur as a racemic mixture (3R/3S), with diverging configuration at the carbon neighbouring the lactone functional group (Strobel, et al., 2002). A similar case is the dihydro isobenzofuran derivative pestacin, which occurs as a racemic mixture of 1R and 1S enantiomers, whereby the chiral carbon C-1 is in -position to the oxygen in the dihydrofuran ring (Harper et al., 2003). Racemization in the case of pestalachloride A, isopestacin and pestacin was proposed to occur after biosynthesis via resonance stabilized cationic

intermediates. Based on structural considerations a similar mechanism may be feasible for 1 and 2, however less favoured since the substituent at C-8 is a methyl group whereas in pestalachloride A (and in a similar fashion in isopestacin and pestacin) an aromatic moiety is located at the respective position. Since racemization does not occur when marilines A or B are exposed to different reaction conditions, it may be concluded that both enantiomers are products of the biosynthetic process and not of post- biosynthetic racemization reactions.

Hypothesis on Biosynthesis Phthalimidine nucleus/Carbon skeleton/Origin of nitrogen:

Compounds 1-4 are phthalimidine derivatives and related to their oxygen-containing counterparts named phthalides, which are more common in nature (Lin et al., 2005). Biosynthetic studies focusing on phthalide structures, e.g. for mycophenolic acid (Bedford et al., 1973) or for phthalide-like metabolites isolated from Talaromyces flavus (Ayer and Racok, 1990) were previously performed by means of feeding experiments with labeled precursors, evidencing the tetraketide nature of the phthalide nucleus. Compounds 1-4, possess the basic skeleton of the tetraketide 3-methylorsellinic acid, however the acetate-derived methyl group in orsellinic acid would be replaced by an ethyl group in the precursor molecules of 1-4 (Geris and Simpson, 2009) (Figure 3). To our knowledge no biosynthetic studies were performed for phthalimidine- like molecules. Phthalimidine biosynthesis, however most probably involves similar polyketide-type reactions as known for phthalides. The most unusual structural characteristic of the phthalimidine derivatives 1-4 is the methyl substituent at C-8. The latter seems to require either, a propionate starter unit (see A in Figure 3) or a methylation (e.g. via a SAM-dependent methyl-transferase) at C-8 (see B in Figure 3). A third possibility would be the loss of a carbon atom from a pentaketide intermediate (not depicted in Figure 3). To our knowledge to date, these types of reaction are not known in fungal polyketide metabolism.

Methylation (see B in Figure 3) would occur most probably at the acetate starter unit, because the methylene carbons in the poly-ß-keto acid would be more prone as the site of attack for a SAM-dependent methyl-transferase than the terminal methyl group of the starter unit (see B in Figure 3). A somewhat similar case represents the myxobacterial

metabolite myxovirescin A, isolated from Myxococcus xanthus, where methylation of the starter unit was also found. In this case feeding studies and the overall organization of the myxovirescin A gene cluster suggested that the biosynthesis of this compound takes place firstly by fusion of an acetate unit with a malonate, followed by methylation, reduction and hydroxylation to produce the starter unit 2-hydroxyvalerianic acid (Simunovic et al., 2006). For the cyanobacterial compound homoanatoxin-a, the incorporation of a SAM-mediated methyl group is suggested to occur in a similar fashion (Namikoshi et al. 2004). Incorporation of an intact propionate starter unit (see A in Figure 3) has been reported for a number of bacterial metabolites, e.g. anthracycline antibiotics such as aclarubicin, or doxorubicin (Fujii and Ebizuka, 1997). Umezawa et al. showed that the common anthracycline intermediate aklavinone, a metabolite isolated from S. galilaeus, is derived from propionyl-CoA and nine malonyl-CoA extender units (Yoshimoto et al., 1981). The best-studied use of a propionate as starter is for daunorubicin from S. peucitus and its C14-hydroxylated derivative doxorubicin from S. coeruleorubicus, which are among the most important antitumor antibiotics in current use (Hutchinson, 1997; Grim et al., 1994). With regard to the biosynthetic origin of propionyl-CoA, it is proposed that the primer is derived from multiple sources such as amino acid catabolism and degradation of odd-numbered fatty acids, but decarboxylation of methylmalonyl- CoA appears to be less likely (Weissman et al., 1998). It appears that the type of reactions probably involved in the formation of marilines, to date was only found in bacteria. Hence, these data open up interesting questions on why biosynthetic steps reported for bacteria are encountered in fungal metabolism. Preliminary studies in our laboratory indicate the presence of bacteria associated with the mycelium of the investigated fungus (data not shown).

The origin of the nitrogen atom of the lactam ring in phthalimidines is probably derived from amino acid metabolism. Based on the structural characteristics in several phthalimidines this seems quite obvious, e.g. staplabin analogues containing serine, phenylalanine, leucine or tryptophan partial structures (Geris and Simpson, 2009). Similarly, the nitrogen precursor of hericenone B and stachybotrin C (Kawagishi et al., 1990; Nozawa et al., 1997) with a phenylethyl moiety connected to the nitrogen atom, is probably phenylalanine or tyrosine.

For marilines A and B (1, 2) it seems likely that 4-hydroxyaniline, produced via chorismic acid and p-amino-benzoic-acid (Sasaoka et al., 1980; Tsuji et al., 1981; Tsuji et al., 1985a; Tsuji et al., 1985b;) (Figure 2 and ppendix – Spectroscopic data supporting information) may be the precursor for the C-12 to C-17 comprising aromatic ring and the nitrogen atom. Incorporation studies of chorismic acid and 4-amonobenzoic acid into the 4-hydroxyaniline moiety of N-(-glutamyl)-4-hydroxyaniline in Agaricus bisporus supports the formation of aniline derivatives in fungi (Tsuji et al., 1985a; Tsuji et al., 1985b). Furthermore, several dihydroxyaniline derivatives are known to be produced in fungal metabolism, e.g. 4-hydroxy-2-methoxyacetanilide isolated from a spruce fungal endophyte and Aspergillus sp. (Findlay et al., 2003) (see depicted structures in Appendix - Spectroscopic data supporting information). Regarding mariline C (3), the N atom may originate from ethylamine (see Figure 3), previously reported to be biosynthetically derived through decarboxylation of alanine (Takeo, 1974; Deng et al., 2008). For mariline D (4), glutamine, glutamate or ammonia may introduce the nitrogen atom.

The carbon skeleton of marilines A and B could be considered unprecedented, due to the existence of the unique extra-methyl group at C-8 amongst all known phthalides/phthalimidines. Feeding experiments are under way in order to determine the building blocks for these most unusual molecules.

A. Methylation in the acetate starter unit hypothesis B. Propionate starter unit hypothesis

O O

SCoA +3xAcetylCoA SCoA + 3x Acetyl CoA

SAM S R Ad Condensation Condensation

O O

O O O O O

SEnz EnzS

Methylation (SAM) O Chorismic acid Cyclisation

PABA

OH Alanine

HO NH2 HOOC OH OH

NH3 or Glutamine or NH Glutamate 2 OH H OH N HN O O HO OH O O O N HO O O

O O N

O O O

Figure 3. Proposed biosynthetic pathways for Mariline A – D.

It is worthwhile to mention that marilines were produced solely on solid biomalt medium supplemented with sea salt, whereas in other media such as Czapek or YPM no phthalimidines were formed. In all four individual culture experiments on biomalt medium supplemented with sea salt, marilines A and B were isolated, whilst marilines C and D were separated only once and in low amounts. The fungus was also not able to grow on biomalt without a sea salt supplement.

Bioactivity

The most interesting activity obtained from the multitude of bioactivity tests was found towards human leukocyte elastase (HLE). This serine protease that has been associated with several pathological conditions including cystic fibrosis, rheumatoid arthritis and infectious diseases (Bieth et al., 1998) HLE is further considered to be the primary source of tissue damage associated with such inflammatory diseases as pulmonary emphysema and adult respiratory distress syndrome (Vender, 1996; Chua and Laurent, 2006; Pham, 2006; Taggart et al., 2005). Small molecular weight HLE inhibitors could be therapeutically useful in the treatment of such diseases, and a number of inhibitors are currently in development (Esper and Martin, 2005; Abbenante and Fairlie, 2005).

Experimental section General Experimental Procedures. See chapter 3 - General methodology. Specifically for marilines A-B, to separate enantiomeric mixtures the HPLC column Lux Cellulose-1 from Phenomenex (250 x 4.5 mm; 5 µm) was used with a flow rate of 1 mL/min (hexane : isopropanol 90:10). The 1H-15N HMBC spectrum was referenced externally to urea (15N chemical shift is reported relative to liquid ammonia).

Fungal material. See chapter 3 - General methodology.

Culture, extraction and isolation. Compounds 1 to 4 were isolated from two different cultures of Stachylidium sp., a first with 12 L and a second with 10 L media volume. Both were performed on an agar-biomalt medium (biomalt 20g/L, 15 g/L agar) supplemented with sea salt during 2 months (12 L culture) and 40 days (10 L culture). An extraction with 5 L EtOAc yielded 5.9 g and 2.1 g of extract, respectively, which were subjected to VLC fractionation in a silica open column using a gradient solvent system with petroleum ether - acetone at 10:1, 5:1, 2:1, 1:1, 100 % acetone and 100 % MeOH, resulting thus 6 VLC fractions for each culture. Compounds 1 and 2 were isolated from both cultures, being hereby described the 12 L culture isolation procedures. Compound 4 was isolated from the first culture (12 L), whilst compound 3 was isolated from the second culture (10 L). Compounds 1 and 2 were isolated from VLC 2, followed by HPLC fractionation using petroleum ether - acetone 11:1 (fraction 2 of 7) and RP-HPLC fractionation using 90 %

MeOH (fraction 3 of 4, 12.6 mg, tR 31 min). The racemic mixture was then separated using a Phenomenex Lux Cellulose-1 HPLC column using hexane : isopropanol 90:10

(Mariline A, 7.2 mg, tR 21 min; Mariline B, 4.8 mg, tR 29 min). Compound 3 was isolated from VLC 4, followed by HPLC fractionation using petroleum ether - acetone 9:2 (fraction 5 of 7, 2.6 mg, tR 48 min) Compound 4 was isolated from VLC 4, followed by HPLC fractionation using petroleum ether - acetone 2:1 (fraction 4 of 7), followed by RP-HPLC with an

Macherey-Nagel Ísis column using 30 % MeOH (fraction 2 of 3, 1.4 mg, tR 7 min).

23 Mariline A (1): colorless oil (600 µg/L 0.12 %); [α] D +14 (c 0.225, acetone); UV

(MeOH) λmax 219 nm (log Є 4.11), 268 (log Є 3.73); IR (ATR) νmax 3330 (br), 2925, 1668, 1605 cm-1; 1H NMR and 13C NMR (Tables 1 and 2); LREIMS m/z 533.3 (M)+; + –4 HREIMS calcd. m/z 533.3141 for C33H43NO5 (M) , found 533.3139; CD (c 2.8 × 10 mol/L, MeOH) λ (ε) = 202 (-9.0), 218 (+12.4), 248 (+6.1), 298 (-3.2).

23 Mariline B (2): colorless oil (400 µg/L, 0.08 %); [α] D -14 (c 0.175, acetone); UV

(MeOH) λmax 219 nm (log Є 4.1), 268 (log Є 3.7); IR (ATR) νmax 3330 (br), 2925, 1668, 1605 cm-1; 1H NMR and 13C NMR (Tables 1 and 2); LREIMS m/z 533.3 (M)+; + –4 HREIMS calcd. m/z 533.3141 for C33H43NO5 (M) , found 533.3139; CD (c 3.1 × 10 mol/L, MeOH) λ (ε) = 202 (+15.8), 218 (-12.2), 248 (-5.2), 293 (+4.3).

Mariline C (3): white amorphous solid (260 µg/L, 0.124 %); UV (MeOH) λmax 207 nm -1 1 (log Є 4.46), 262 nm (log Є 3.95); IR (ATR) νmax 3391 (br), 2926, 1664, 1607 cm ; H NMR and 13C NMR (Tables 1 and 2); LRESIMS m/z 388.5 (M+H)+; HRESIMS calcd. + m/z 410.2302 for C23H33NO4Na (M+Na) , found 410.2290; no CD spectra revealed the presence of a racemic mixture.

Mariline D (4): amorphous solid (108 µg/L, 0.022 %); UV (MeOH) λmax 216 nm (log Є -1 1 3.90), 259 nm (log Є 3.71); IR (ATR) νmax 3346 (br), 2926, 1670, 1610 cm ; H NMR and 13C NMR (Tables 1 and 2); LRESIMS m/z 206.0 (M-H)-, m/z 207.9 (M+H)+; + HRESIMS calcd. m/z 230.0788 for C11H13NO3Na (M+Na) , found 230.0786; no CD spectra revealed the presence of a racemic mixture.

Racemization experiments. Racemization experiments were based on literature data (Stuck et al., 2003)., where racemization of a phthalimidine nucleus was achieved with very low acidic/alkaline concentrations (0.0005 M KOH). Each experiment contained

200 µg of mariline A dried on a stream of N2 in a glass vial which was suspended in 400 µL solution with the following conditions: a) water, b) 0.0005 M KOH, c) 0.0025 M KOH, d) 0.5 M KOH, e) 0.0005 N HCl, d) 0.5 N HCl. 200 µg mariline B were also tested at the range of 0.5 N KOH. All samples were then incubated in a water bath at 55 ºC for 2 hours, after which they were neutralized with the same molar proportion of acid/alkaline solution. After drying the sample in gaseous nitrogen the samples were injected in a HPLC chiral phase column with the same solvent system used to separate the enantiomers marilines A and B for detection of racemization (see General Experimental Procedures).

Biological activity assays. See chapter 3 - General methodology.

5. Marilones A – J, Unusual Phthalides from the Sponge-derived Fungus Stachylidium sp.

Abstract The marine-derived fungus Stachylidium sp. was isolated from the sponge Callyspongia sp. cf. C. flammea. Culture on a biomalt medium supplemented with sea salt led to the isolation of eight new phthalide derivatives with unusual structural motives, i.e. marilones A – J (1-8, 10, 11), and the known compound silvaticol (9). In the epimeric compounds 10 and 11 the phthalide (=isobenzofuranone) nucleus is modified to an isobenzofuran ring with ketal and acetal functionality. The skeleton of marilones A, B, D and F-J is most unusual, and its biosynthesis is suggested to require unusual biochemical reactions considering fungal secondary metabolism. Marilone A (1) was found to have antiplasmodial activity against Plasmodium berghei with an IC50 of 12.1 µM. Marilone B (2) showed selective antagonistic activity towards the serotonin receptor 5HT2B with a Ki value of 7.7 µM.

11 O 88O O R R O 5 1 O 2 1 3 10 10 O O O 9 9

3' 8' 3,R = 3' 8' 1,R1 = 2 5' 1' 5' 1' 9' 10' 9' 10'

9,R=H 2,R1 =H 2

12 8 6 8 5 OH OH HO HO

11 10 10 O O O O 11 11 9 9

4 5

16 13 11 OH

57 1 9 18 OH O O 17 6

7 O 8 OH 6 OH 9 O 1 11 8 HO 1 O O 10 7 8

11 O 6 O 12 8 O 1' 8 O O O 13 10' O 1 10 O 10 H O H O 9 9 10 11 Figure 1. Secondary metabolites 1 – 11 isolated from Stachylidium sp.

Introduction

Phthalides are a class of structurally most diverse secondary metabolites with more than 180 naturally occurring compounds described (Lin et al., 2005). They are produced by a wide range of organisms, i.e. marine and terrestrial fungi belonging to genera such as Aschochyta (Seibert et al., 2006), Aspergillus (Fujita et al., 1984; Achenbach et al., 1985), Alternaria (Suemitsu et al., 1995; Kawahara et al., 1988), Penicillium (Smith, 1952), Hericium (Kawagishi et al., 1990) or Talaromyces (Ayer and Racok, 1990), but also by plants and liverworts (Lin et al., 2005). Phthalides exhibit an equally broad spectrum of bioactivity, including modulation of the central nervous system, protection against brain eschemia, modulation of platelet aggregation and cardiac function, inhibition of smooth muscle cell proliferation, anti- angina activity, smooth muscle relaxation, as well as antibacterial, antifungal, antiviral and phytotoxic activity (Lin et al., 2005). The medically most important member of this family of natural products is mycophenolic acid, firstly isolated from Penicillium brevicompactum, and used in form of its derivative mycophenolate mofetil as an immunosupressant drug (Dewick, 2009).

During our search for new natural products produced from the marine-derived fungus Stachylidium sp., several structurally unusual phthalide derivatives, i.e. marilones A - J, were isolated from a culture on agar-BMS media supplemented with artificial sea salt (Scheme 1). Albeit phthalide-like structures are not rare, the structural skeleton of marilones A, B, D, F - J is most unusual, and its biosynthesis is suggested to require unique reactions in fungal secondary metabolism. Marilone A (1) exhibited antiplasmodial activity against Plasmodium berghei with an IC50 of 12.1 µM. Marilone

B (2) showed a specific antagonistic effect on the serotonin receptor 5HT2B with a Ki value of 7.7 µM.

Results

The molecular formula of 1, was deduced by accurate mass measurement (HREIMS) to 13 be C21H28O4, requiring eight degrees of unsaturation. The C NMR and DEPT135 spectra contained 21 carbon resonances, including six resulting from methyl groups,

three from sp2 methines, one from a sp3 methine, whereas further three signals resulted from methylene groups and eight resonances were assigned to quaternary carbons (Tables 1, 2 and Appendix – Complete 2D NMR data). The 1H NMR spectrum of 1 displayed a singlet resonance for the aromatic methine (6-CH) at δ 6.95 indicating together with UV and 13C NMR data the presence of a penta-substituted benzene ring.

The methyl group 10-CH3 (δC 8.8) was linked to C-4 of the aromatic ring due to heteronuclear long range correlations of the methyl protons with C-3, C-4 and C-5. The methoxyl group 9-OCH3 (δH 3.98) had a heteronuclear long range correlation to C-3 of the aromatic ring, thus clearly delineating it’s position. Besides the aromatic proton 6-H, the 1H NMR spectrum showed two further resonance signals in the downfield shifted 13 1 13 region (δH 5.52 and 5.09) indicating together with C NMR and H- C HMBC data the presence of a geranyl substituent. The C-1’ to C-10’ part of the molecule was deduced from two proton coupling spin systems observed in the 1H-1H COSY spectrum, namely 1 13 1’-H2 to 2’-H (J = 6.6 Hz) and 4’-H2 to 6’-H through 5’-H2. H- C HMBC data showed correlations from 9’-H3 to C-2’, C-3’ and C-4’, and from methyl protons 8’-H3 and 10’-

H3 to C-6’and C-7’, disclosing a geranyl fragment. Based on literature comparisons we established the configuration of 2’/3’ as E (Tanaka et al., 1985). The aromatic quaternary carbon C-5 (δC 164.2) had a carbon resonance indicating a connection to an oxygen atom. The monoterpenyl substituent was established to be connected to C-5 through an oxygen atom due to heteronuclear long range correlations of 1’-H2 (δH 4.69, 4.74) to C-5. The 1H-13C HMBC spectrum exhibited a correlation from 6-H to C-8. Furthermore, the 13C NMR resonance of C-8 at δ 77.0 was found to be characteristic for a carbon bound 1 1 to an oxygen atom. The H- H COSY spectrum showed a coupling of 8-H with 11-H3 (J = 6.6 Hz), and the 1H-13C HMBC spectrum contained correlations from 8-H to C-6, C-7 and C-2 of the penta-substituted aromatic ring, as well as to the carbonyl carbon C-1. Ring double bond equivalents required a second ring within compound 1, and together with heteronuclear correlations of 8-H to C-1 and the carbon resonance of C-1 at δC

168.2 indicating the presence of a carbonyl group, gave evidence for a C-8-methylated phthalide skeleton, i.e. the C-1 to C-11 part of the structure. Since the resonance signal for 6-H did not show heteronuclear long range correlations to that of the carbonyl C-1, but correlated with the sp3 methine C-8, the carbonyl group was assigned at C-1. This way, a phthalide-nucleus identical to that of the known natural product nidulol was formed.[6] To further prove that the carbonyl group is positioned at C-1 and not at C-8 of

1, 1H NMR spectra of 1 were compared with those of nidulol, silvaticol (9) and derivatives (see Appendix – Spectroscopic data supporting information). The latter are known regioisomeric phthalides with the carbonyl group at C-1 and C-8, respectively. 1 Differences in H NMR resonances can be discerned especially for 6-H, resonating at δH

6.59 (CDCl3) for nidulol and δH 7.04 (CDCl3) for silvaticol (Kawahara et al., 1988). The 1 H NMR spectrum of 1 (δH 6.54 in CDCl3) was shown to be similar to that of nidulol and the nidulol derivative, 5-(3',3'-dimethylallyloxy-7-methoxy-6-methylphthalide with

6-H resonating at δH 6.62 (see Appendix – Spectroscopic data supporting information and Suemitsu et al., 1995). For compound 1 the trivial name marilone A is suggested.

The molecular formula of 2 was deduced by accurate mass measurement (HREIMS) to be C11H12O4, requiring six sites of unsaturation. The NMR spectral data (see Tables 1, 2 and Appendix – Complete 2D NMR data) indicated that compound 2 is identical to 1, except for the missing geranyl moiety attached to the hydroxy group at C-5. We propose the trivial name marilone B for compound 2.

The molecular formula of 3, was deduced by accurate mass measurement (HREIMS) to be C20H26O4, requiring eight degrees of unsaturation. The spectroscopic data of 3 revealed that the compound is very similar to 1 (Tables 1, 2 and Appendix – complete 2D NMR data). In contrast to compound 1, however, resonance signals for a methylene group, i.e. 1-CH2 (δC 68.9) were found in the NMR spectra, instead of those for a methine (8-CH) and methyl group (11-CH3) as in 1. The resonance signal for 6-H did not have a heteronuclear long range correlations to C-1, but correlated with the carbonyl carbon C-8. Hence, the carbonyl group in 3 was assigned to be located at C-8, thus, forming a phthalide-nucleus as present in silvaticol (9). For compound 3, the name marilone C is suggested.

The molecular formula of 4, was deduced by accurate mass measurement (HRESIMS) to be C12H18O4, requiring four sites of unsaturation. The NMR spectroscopic data (Tables 4, 5 and Appendix – complete 2D NMR data) indicated again a penta- substituted benzene ring identical to that in 1-3. A 1H NMR coupling from the methine 1 8-H to 12-H3 (J = 6.3 Hz) was discerned in the H NMR spectrum, whereas C-8 had a carbon shift (δC 65.9) characteristic for oxygen substitution. The methoxyl function 11-

OCH3 was connected to the methylene 1-CH2 as deduced from heteronuclear long range

correlations of the methoxyl protons to C-1. Compound 4 is thus a monocyclic compound, for which we propose the trivial name marilone D.

The molecular formula of 5, was deduced by accurate mass measurement (HRESIMS) to be C11H16O4, requiring four sites of unsaturation. The structure of 5 was found to be almost identical to that of 4 (Tables 4 and 5 and Appendix – complete 2D NMR data). The only difference between them was discerned to be the substitution of C-8. In case of compound 4 this carbon was substituted with the methyl group (12-CH3), whereas in compound 5 C-8 was an oxygenated methylene group. Consequently, the 1H NMR spectrum of 5 exhibited a singlet resonance for a methylene group at δH 4.64. We propose the trivial name marilone E for compound 5.

The molecular formula of 6 was deduced by accurate mass measurement (HRESIMS) to 13 be C18H28O4, requiring five sites of unsaturation. The C NMR and DEPT135 spectra indicate the presence of 18 resonances, including five for methyl, four sp3 methylene, one sp2 methine, two sp3 methine groups and six for quaternary carbons (see Table 3). Compound 6 has the close to identical penta-substituted aromatic ring as found for 1-5 (notice: carbons/hydrogens have different number assignment), as proven by 1H-13C HMBC correlations from 12-H to C-8, C-10, C-11, C-13 and C-14. The methyl group 3 15-CH3 was attached to the sp methylene 14-CH2, completing the substituent at C-13. Based on 1H-13C HMBC and 1H-1H COSY correlations we found that the aromatic substituent at C-8 was an aliphatic chain giving rise to two 1H-1H spin systems, 1 1 interrupted by a ketone functionality (C-5, δC 214.8). The H- H COSY spectrum revealed a spin system ranging from 1-H3 to 4-H2, including the hydroxylated 3-H (δH

3.85), and from 6-H to 16-H3 and 7-H2. 4-H2, 6-H, 16-H3 and 7-H2 displayed heteronuclear long range correlations to the ketone carbon C-5 and thus connected the partial structures that were deduced from 1H coupling data. Heteronuclear long range correlations also cleared the connection of 7-CH2 to C-8 of the aromatic ring. For compound 6 the name marilone F is suggested.

The molecular formula of 7 was deduced by accurate mass measurement (HRESIMS) to be C11H12O4, requiring six sites of unsaturation. Spectroscopic data clearly suggested, that compound 7 shares the penta-substituted aromatic ring with compounds 1-6. The sp3 methine 1-CH displayed a 13C NMR resonance signal typical for a carbon bound to

1 1 oxygen (δC 67.1) and showed H- H coupling with 9-H2 (J = 6.5 and 1.6 Hz). 1-H had heteronuclear long range correlations with C-2, C-3 and C-7 of the aromatic ring, suggesting that it is connected to C-2 of the aromatic moiety. All of the protons, 1-H, 6-

H and 9-H2 had heteronuclear long range couplings to a carbonyl carbon (C-8, δC 206.4), which is thus connected to C-7 of the aromatic ring. Thus, in 7 the second ring is not heterocyclic, and cyclisation during biosynthesis probably involved the C-11 methyl carbon of 1 to give the methylene group 9-CH2 in 7. For compound 7 the name marilone G is suggested.

The molecular formula of 8 was deduced by accurate mass measurement (HREIMS) to 13 be C8H10O3, requiring four sites of unsaturation. The C NMR and DEPT135 spectra included eight resonances, i.e. two for methyl, one for a sp2 methine, and one further resonance for a sp3 methylene group. Further four resonances were due to quaternary carbons, which thus made up half of the carbons encountered in the molecule (see compound spectroscopic data). The methyl protons 7-H3 coupled with 6-H2 (J = 7.5 Hz) as evident from 1H NMR data, and both had heteronuclear long range correlations with

C-5, the latter being a quaternary carbon connected to oxygen (δC 164.9). The methyl group 8-CH3 had heteronuclear long range correlations with C-1, C-2 and C-3, and was assigned at C-2 (δC 98.2). C-3 was characterized by a downfield shifted carbon resonance, which indicated it to be connected to oxygen (δC 165.6). The HMBC correlations of 4-H and 8-H3 to C-3 placed this hydroxylated quaternary carbon between

C-2 and C-4. The quaternary carbon C-1 (δC 166.0) was identified as being part of a carboxylic functionality, also evidenced by an IR absorption at 1645 cm-1. Consideration of the molecular formula required compound 8 to be monocyclic. Thus, C-1 was connected to C-5 through an oxygen atom forming a six-membered lactone ring. We propose the trivial name marilone H for compound 8, which is already described as a synthetic compound (Suzuki et al., 1973).

Spectroscopic data of 9 were determined to be identical to those of silvaticol (see Appendix – Spectroscopic data supporting information and Kawahara et al., 1988).

The molecular formulae of 10 and 11 were deduced by accurate mass measurement

(HREIMS) and found to be identical, i.e. C23H34O5, requiring seven degrees of 13 unsaturation. The C NMR and DEPT135 spectra contained 23 carbon resonances,

including eight resulting from methyl, three from sp2 methines, one from a sp3 methine and three from methylene groups, whereas eight resonances were assigned to quaternary carbons. 1D and 2D NMR spectroscopic data of both compounds revealed that the penta-substituted aromatic ring and the substituents at C-3, C-4 and C-5 were identical to those in 1 and 3 (Tables 1, 2 andAppendix – Complete 2D NMR data). Compounds 10 and 11 have the same planar structure, but differ in stereochemistry. For clarity in the description of the structure elucidation, only the spectroscopic data of 10 will be described initially. The NMR spectra revealed signals for three methoxyl groups

9-OCH3, 12-OCH3 and 13-OCH3 (δC at 59.8, 49.7 and 54.2, respectively).

Heteronuclear long range correlations from 12-H3 and 13-H3 to C-8 and C-1, respectively allowed to locate the methoxyl moieties at the respective carbons. The carbons C-1 (δC at 105.4) and C-8 (δC at 111.4) displayed strongly downfield shifted resonances for sp3 carbons, indicating that both of them were connected to two oxygen atoms. The methine proton 1-H, had heteronuclear long range correlations to C-3, C-7 and C-8, making the presence of a furan ring with a ketal and an acetale functionality most likely.

HMBC couplings from 11-H3 with C-7 and C-8 connected this methyl group to C-8. Differences between 10 and 11 were established to be related to the configuration at C- 8. This was evident from different NOE interactions. Namely, in 10 the protons of the methyl group 11-CH3 had a NOE correlation with 13-OCH3, whilst 12-OCH3 at C-8 had a NOE correlation with 1-H. In 11 the NOE correlations were the opposite, i.e. 11-CH3 correlates with 1-H and 13-OCH3 correlates with 12-OCH3 (see Figure 2). Initially, it seemed likely, that compounds 10 and 11 are artifacts of compound 1, probably produced during extraction and isolation by methanolysis. However, heating of compound 1 for two hours at 50 ºC in methanol did not produce 10 and 11. For compounds 10 and 11 the names marilone I and J are suggested, respectively.

O O O O O O

O O H O H O

(3) (4)

Figure 2. Key NOE correlations for compounds 10 and11

Table 1. 13C-NMR spectroscopic data for compounds 1, 2, 3, 10 and 11.

1 3 10 11 2 a, b a, b a, b a, b a, b position δC, mult. δC, mult. δC, mult. δC, mult. δC, mult.

1 168.2, qC 68.9, CH2 105.4, CH 104.8, CH 168.2, qC 2 110.0, qC 128.4, qC 121.4, qC 121.5, qC 109.1, qC 3 158.0, qC 153.9, qC 154.8, qC 154.9, qC 158.9, qC 4 120.4, qC 124.8, qC 119.0, qC 119.2, qC 118.8, qC 5 164.2, qC 159.7, qC 160.4, qC 160.4, qC 163.2, qC 6 100.8, CH 102.0, CH 100.2, CH 100.2, CH 103.6, CH 7 154.2, qC 125.8, qC 141.7, qC 141.3, qC 153.9, qC 8 77.0, CH 171.1, qC 111.4, qC 110.8, qC 76.6, CH

9 62.1, CH3 59.3, CH3 59.8, CH3 59.8, CH3 62.0, CH3

10 8.8, CH3 9.8, CH3 9.3, CH3 9.3, CH3 8.6, CH3

11 20.9, CH3 28.1, CH3 27.8, CH3 21.0, CH3 12 49.7, CH3 50.5, CH3 13 54.2, CH3 55.5, CH3

1' 66.5, CH2 66.4, CH2 66.1, CH2 66.2, CH2 2' 120.1, CH 120.5, CH 120.8, CH 120.9, CH 3' 142.1, qC 141.6, qC 141.1, qC 141.1, qC

4' 40.1, CH2 40.1, CH2 40.1, CH2 40.1, CH2

5' 26.9, CH2 26.9, CH2 27.0, CH2 27.0, CH2 6' 124.6, CH 124.6, CH 124.6, CH 124.7, CH 7' 132.1, qC 132.1, qC 132.0, qC 132.0, qC

8' 25.8, CH3 25.8, CH3 25.8, CH3 25.8, CH3

9' 16.7, CH3 16.7, CH3 16.6, CH3 16.7, CH3

10' 17.7, CH3 17.7, CH3 17.7, CH3 17.7, CH3 a b Acetone-d6 (1, 2, 3, 10, 11), 75.5 MHz. Implied multiplicities determined by DEPT.

Table 2. 1H-NMR spectroscopic data for compounds 1, 2, 3, 10 and 11.

1 3 10 11 2 a, b a, b a, b a, b a, b position δH (J in Hz) δH (J in Hz) δH (J in Hz) δH (J in Hz) δH (J in Hz) 1 5.50, s 6.32, s 6.02, s 2 3 4 5 6 6.95, s 7.03, s 6.60, s 6.59, s 6.75, s 7 8 5.43, q (6.6) 5.37, q (6.6) 9 3.98, s 3.96, s 3.90, s 3.89, s 3.98, s 10 2.09, s 2.15, s 2.07, s 2.06, s 2.10, s 11 1.54, d (6.6) 1.62, s 1.55, s 1.48, d (6.6) 12 2.85, s 2.94, s 13 3.38, s 3.50, s 1' a: 4.69, dd (6.6, 12.1) 4.71, d (6.6) 4.64, d (6.6) 4.63, d (6.6) b: 4.74, dd (6.6, 12.1) 2' 5.52, t (6.6) 5.51, t (6.6) 5.48, t (6.6) 5.48, t (6.6) 3' 4' 2.10, m 2.10, m 2.08, m 2.07, m 5' 2.13, m 2.14, m 2.11, m 2.11, m 6' 5.09, m 5.10, m 5.11, m 5.10, m 7' 8' 1.63, br s 1.63, br s 1.64, br s 1.64, br s 9' 1.76, br s 1.78, br s 1.75, br s 1.75, br s 10' 1.58, br s 1.58, br s 1.59, br s 1.58, br s a b Acetone-d6 (1, 2, 3, 10, 11), 300 MHz. Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY).

Table 3. 1D and 2D NMR spectroscopic data for compound 6

a, b, e a, b a, c a, d position δC, mult. δH (J in Hz) COSY HMBC

1 10.1, CH3 0.85, t (7.3) 2 2, 3

2 30.6, CH2 1.35, m 1, 3 1, 3 3 69.4, CH 3.85, m 2, 4 1

4 49.5, CH2 a :2.41, dd (4.4, 16.5) 3, 4b 2, 3, 5 b: 2.51, m 4a 2, 3, 5 5 214.8, qC 6 48.1, CH 2.87, m 7a, 7b, 16 5, 7, 8, 16

7 29.8, CH2 a: 2.81, m 6, 7b 5, 6, 8, 9, 13, 16 b: 2.53, m 6, 7a 5, 6, 8, 9, 13, 16 8 122.1, qC 9 159.2, qC 10 115.6, qC 11 155.6, qC 12 111.5, CH 6.51, s 8, 10, 11, 14 13 141.9, qC

14 26.3, CH2 2.52, m 15 8, 12, 13, 15

15 15.8, CH3 1.10, t (7.5) 14 13, 14

16 15.9, CH3 0.94, d (6.7) 6 5, 6, 7

17 60.4, CH3 3.65, s 9

18 9.4, CH3 2.08, s 9, 10, 11 a b Acetone-d6, 300/75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT.

Table 4. 13C-NMR spectroscopic data for compounds 4 and 5

4 5 a, b a, b position δC, mult. δC, mult.

1 66.1, CH2 66.9, CH2 2 119.6, qC 120.3, qC 3 159.3, qC 160.0, qC 4 116.1, qC 117.5, qC 5 157.1, qC 157.9, qC 6 108.5, CH 111.6, CH 7 147.2, qC 141.4, qC

8 65.9, CH 62.6, CH2

9 61.8, CH3 62.2, CH3

10 9.1, CH3 9.2, CH3

11 57.8, CH3 58.1, CH3

12 25.4, CH3 a b Acetone-d6 (4), MeOD (5), 75.5 MHz. Implied multiplicities determined by DEPT.

Table 5. 1H-NMR spectroscopic data for compounds 4 and 5 4 5 a a position δH (J in Hz) δH (J in Hz) 1 a: 4.39, d (9.9) 4.53, s b: 4.43, d (9.9) 2 3 4 5 6 6.91, s 6.77, s 7 8 5.06, q (6.3) 4.64, s 9 3.67, s 3.74, s 10 2.09, s 2.15, s 11 3.32, s 3.42, s 12 1.33, d (6.3) a Acetone-d6 (4), MeOD (5), 300 MHz.

Stereochemistry

Compounds 1, 2 and 4 possess a single chiral center at C-8. The measurement of the specific optical rotation for these compounds yielded values close to zero, and furthermore, the CD measurements showed hardly any CD effect for the referred compounds. The latter was expected at around 260 nm due to the proximity of the chiral center to the chromophoric penta-substituted benzene ring. We thus assumed the presence of racemic mixtures for these chiral compounds. Extensive trials to separate the enantiomers employing three different HPLC chiral stationary phases were performed, but unsuccessful. However, the presence of racemic mixtures was proven for the analogous, nitrogen-containing compounds, i.e. pththalimidine derivatives isolated from the same fungus (Almeida et al., unpublished data). The chiral centre C-1 in compound 7 also seems to be present in R and S configuration, since measurements of optical rotation yielded values close to zero.

Biological activity

Marilones A, B and C (1-3) were tested for antiplasmodial activity, and marilone A exhibited an IC50 of 12.1 µM against Plasmodium berghei (see Appendix – Bioactivity results). Interestingly, marilone C (3) showed no activity at the 25 µM level, indicating that the methyl group 11-CH3 and/or the position of the ketone functionality is essential for this bioactivity. Marilones A, B, C, D (1-4) and I (10) were tested for cytotoxic activity toward three cancer cell lines (NCI-H460, MCF7 and SF268). Marilone A and C (1, 3) showed weak antiproliferative activity with an average GI50 of 36.7 and 26.6 µM, respectively (seeAppendix – Bioactivity results). Marilone B (2) and H (8) were assayed in a panel of 44 psychoactive receptors, including 11 serotonin receptors, and marilone B showed specific antagonistic effect on the serotonin receptor 5HT2B with a Ki value of 7.7 µM. Marilones A, B and C were tested in protein kinases (DYRK1A and CDK5) inhibition activity assay, inhibition of the viral HIV-1 and HIV-2 induced cytopathogenic effect in MT-4 cells, in antibacterial (Escherichia coli, Bacillus megaterium), antifungal (Mycotypha microspora, Eurotium rubrum, and Microbotryum violaceum), and antialgal (Chlorella fusca) assays at the 50 µg/mL disc level, they were evaluated for

inhibition of the following panel of proteases: Chymotrypsin, trypsin, the protease elastase HLE, papain, porcine cease and acetylcholine esterase (tested at the 100 µM level) but exhibited no activity in any of these tests. Marilones A and C were also evaluated against 3-T3-L1 murine adipocytes assay and exhibited no activity. Marilones A and B were evaluated against three sub-types of Influenza A virus (H1N1, H5N1 and H3N2), Influenza B virus (Flu B) and exhibited no activity. Marilone A was evaluated against the Severe Acute Respiratory Syndrome coronavirus (SARS) and Hepatitis B virus but did not show any activity. Marilone B was evaluated against the Herpes Simplex Virus-2 (HSV-2) and the Respiratory Syncytial virus (RSV) and exhibited no activity. Marilones A and D were further tested against two antibiotic resistant Mycobacterium tuberculosis strains, MTB72 and R46 (tested at the 50 µg/mL disc level), but did not show any relevant activity. Marilones C, G and H were tested in a panel of anti-diabetic activity assays: PPAR ligand assay, PPAR co-activator assay, PTP-1B inhibitor assay and glucose uptake assay (both 3T3-l1 cells and primary rat/mouse adipocytes, tested at the 100 µM dose), but did not show any activity. Marilones C, G, H and E were tested for the inhibition of the NF-kB protein complex assay (at the 100 µM), but did not show any activity.

Discussion

Similar molecules and biosynthesis Phthalide derivatives are compounds of the polyketide metabolism, which are common in nature (Lin et al., 2005). Secondary metabolites 1 and 2 discovered in the marine- derived Stachylidium sp. were found to be derivatives of the natural product nidulol, whilst compound 3 was a derivative of silvaticol (9) (see Appendix – Spectroscopic data supporting information), formerly described from the fungus Aspergillus silvaticus (Kawahara et al., 1988). Nidulol and silvaticol (9) are regioisomeric compounds and differ in terms of the position of the carbonyl group, which either is placed peri to the aromatic hydrogen as in 3 and 9, or it is peri positioned to the aromatic methoxyl moiety, e.g. in 1 and 2. The here investigated Stachylidium species is thus able to

produce both types of phthalides, which are suspected to differ significantly in terms of their biosynthesis (Figure 3). Whereas compound 3 is simply the O-prenylated form of silvaticol (9), the nidulol derivatives 1 and 2 are distinguished by an additional methyl substituent (11-CH3) at C- 8. In terms of biosynthesis, i.e. polyketide metabolism, this substitution is most unusual and unique amongst all the known phthalides. The non-phthalide compounds of the Stachylydium metabolites, i.e. 4, 6-8, also share this structural characteristic of an additional carbon atom (C-12 in 4, C-15 in 6, C-9 in 7, C-7 in 8), which is hard to explain by the usual fungal polyketide metabolism.

Biosynthetic studies focusing on phthalide structures, e.g. for mycophenolic acid (Bedford et al. 1973), nidulol and silvaticol (Ayer and Racok, 1990), were previously performed by means of feeding experiments with labeled precursors, evidencing the tetraketide nature of the phthalide nucleus (Figure 2). Compounds 1-7, possess a basic skeleton which is related to that of the well known tetraketide 3-methyl-orsellinic acid (Geris and Simpson, 2009). Closing of the lactone ring would for compounds 1 and 2 require the oxidation of C-8 to obtain a hydroxy group, which subsequently could form a lactone with the C-1 carboxyl group (Figure 3A). In contrast to that for 3 and 9, a reduction of the C-1 carboxyl group to an alcoholic function and an oxidation of C-8 to a carboxylic function would be required (Figure 3B).

Most intriguing however is, that in compounds 1, 2, 4, 6 and probably also 7 the acetate- derived methyl group 8-CH3 in the methyl orsellinic acid precursor would be replaced by an ethyl group (Figure 3). Thus, biosynthesis seems to require either, a propionate starter unit (see C in Figure 3) or a methylation (e.g. via a SAM-dependent methyl- transferase) at C-8 (see D in Figure 3). A third possibility would be the loss of a carbon atom from a pentaketide intermediate (not depicted in Figure 3). To our knowledge, to date these types of reactions are not known in fungal polyketide metabolism. Feeding experiments are under way in order to determine the building blocks for these most unusual molecules.

OH OH OH OH OH OH

O O O

HO HO HO 3-methylorsellinic acid ABC D

OH OH OH OH O O O O O O O OH OH HO HO RO RO O 1 , 2 1 , 2

OH O OH

O O HO HO O = methylation via SAM

= acetate unit

O O O = propionate unit

O O HO HO O nidulol silvaticol

Figure 3. Tetraketide nature of the phthalides nidulol (A) and silvaticol (B), and hypothetical biosynthetic pathways (C, D) for marilones A and B (1, 2).

It is worthwhile to mention that marilones were produced solely on solid biomalt medium (BMS) supplemented with sea salt, whereas in other media such as Czapek or YPM no phthalides were formed.

Experimental Section General Experimental Procedures. See chapter 3 - General methodology. Fungal material. As described in chapter 3 - General methodology.

Cultivation, extraction and isolation. Compounds 1-9 were isolated from a 60 days culture (12 L) of Stachylidium sp. on an agar-biomalt medium supplemented with sea salt (BMS). An extraction with 5 L EtOAc yielded 5.9 g of extract which was subjected to a VLC fractionation in an open column using silica as solid phase and a gradient solvent system with petroleum ether - acetone of 10:1, 5:1, 2:1, 1:1, 100 % acetone and 100 % MeOH, resulting in six VLC fractions. Compounds 10 and 11 were isolated from a second 10 L culture on BMS (cultivation time: 60 days), from which 1.0 g extract was obtained. Compounds 1 and 3 were isolated from VLC fraction 1. VLC fraction 1 was fractionated using petroleum ether – acetone 90:1 and 10:1 in order to eliminate fatty acid content of the sample. The VLC fraction 10:1 was subjected to NP-HPLC fractionation using petroleum ether – acetone 30:1 to yield a mixture of both compounds (sub-fraction 4 of 7). Further fractionation using MeOH - H2O 8:2 (RP-

HPLC, Isis column) yielded compound 1 (sub-fraction 1 of 2; 19 mg, tR 13 min) and compound 3 (sub-fraction 2 of 2; 14.2 mg, tR 15 min). Compounds 2 and 9 were isolated from VLC fraction 2, followed by NP-HPLC fractionation using PE-acetone 11:1 to yield a mixture of both compounds (fraction 6 of

7). Further fractionation using 40 % MeOH / 60 % H2O (RP-HPLC, Isis column) yielded compound 2 (fraction 1 of 2; 5.2 mg, tR 35 min) and the know compound 9, silvaticol (fraction 2 of 2; 5.6 mg, tR 38 min). Compound 4 was isolated from VLC fraction 3, followed by NP-HPLC fractionation using petroleum ether - acetone 5:1 (fraction 4 of 10). This sub-fraction was further purified using 50 % MeOH / 50 % H2O (RP-HPLC, Isis column; fraction 2 of 4, 4.2 mg, tR 10 min). Compound 5 was isolated from VLC fraction 3, followed by NP-HPLC fractionation using petroleum ether - acetone 5:1 (fraction 7 of 10). This sub-fraction was further purified using 50 % MeOH / 50 % H2O (RP-HPLC, Isis column; fraction 2 of 4, 4 mg, tR 3 min). Compound 6 was isolated from VLC fraction 2, followed by NP-HPLC fractionation using petroleum ether - acetone 11:1 (fraction 3 of 7). This sample was further fractionated with 90 % MeOH / 10 % H2O (RP-HPLC, Isis column, fraction 2 of 4, 1.5 mg, tR 15 min) Compound 7 was isolated from VLC fraction 3, followed by NP-HPLC fractionation using petroleum ether - acetone 5:1 (fraction 9 of 10). This sub-fraction was further

purified using 50 % MeOH / 50 % H2O (RP-HPLC, Isis column; fraction 1 of 4).

Finally we reached purification using 30 % MeOH / 70 % H2O (RP-HPLC, Isis column, fraction 6 of 6, 2.5 mg, tR 14 min). Compound 8 was isolated from VLC fraction 3, followed by NP-HPLC fractionation using petroleum ether - acetone 5:1 (fraction 3 of 10). This sub-fraction was further separated using petroleum ether - acetone 13:1 (fraction 3 of 4), which was finally purified with 40 % MeOH / 60 % H2O using RP-HPLC (fraction 2 of 2, 5.8 mg, tR 12 min). Compounds 10 and 11 were isolated in a second culture in the same procedures as for compounds 1 and 2, except the last RP-HPLC fractionation which was performed with

85 % MeOH /15 % H2O (compound 10, sub-fraction 1 of 2; 1.6 mg, tR 9 min and compound 11, sub-fraction 2 of 2; 1.7 mg, tR 11 min).

In two other cultures of the same fungus in the same medium (2 months; 40 days), only marilone A and marilone C were detected (both in quantities lower than 2.5 mg).

Marilone A (1): transparent oil (1.6 mg/L, 0.32 %); UV λmax (MeOH)/nm: 219 (log  -1 1 13 4.38), 261 (log  2.95); IR (ATR) νmax 2964, 1745, 1603 cm ; H NMR and C NMR + + (Tables 1 and 2); LREIMS m/z 344.2 (M) ; HREIMS calcd. for C21H28O4 (M) m/z 344.1988, found 344.1996.

Marilone B (2): white amorphous solid (0.4 mg/L, 0.09 %); UV λmax (MeOH)/nm: 215 -1 1 (log  4.09), 260 (log  2.83); IR (ATR) νmax 3238 (br), 2931, 1708, 1601 cm ; H NMR and 13C NMR (Tables 1 and 2); LREIMS m/z 208.1 (M)+; HREIMS calcd. for + C11H12O4 (M) m/z 208.0736, found 208.0737.

Marilone C (3): transparent oil (1.3 mg/L, 0.27 %); UV λmax (MeOH)/nm: 223 (log  -1 1 13 4.10), 255 (log  2.80); IR (ATR) νmax 2921, 1760, 1619 cm ; H NMR and C NMR + + (Tables 1 and 2); LREIMS m/z 330.2 (M) ; HREIMS calcd. for C20H26O4 (M) m/z 330.1831, found 330.1833.

Marilone D (4): white amorphous solid (350 µg/L, 0.068 %); UV λmax (MeOH)/nm:

212 (log  4.04), 226 (log  3.82), 271 (log  3.27); IR (ATR) νmax 3338 (br), 2929, 1610, 1458, 1414 cm-1; 1H NMR and 13C NMR (Tables 3 and 4); LRESIMS m/z 224.9 - + (M-H) ; HRESIMS calcd. for C12H18O4Na (M+Na) m/z 249.1097, found 249.1101.

Marilone E (5): white amorphous solid (333 µg/L, 0.068 %); UV λmax (MeOH)/nm:

210 (log  4.25), 229 (log 3.94), 276 (log  3.18); IR (ATR) νmax 3347 (br), 2929, 1602, 1457, 1418 cm-1; 1H NMR and 13C NMR (Tables 3 and 4); LRESIMS m/z 211.1 - + (M-H) ; HRESIMS calcd. for C11H16O4Na (M+Na) m/z 235.0941, found 235.0948.

23 Marilone F (6): white amorphous solid (125 µg/L, 0.025 %); [α]D -33 (c 0.1, MeOH);

UV λmax (MeOH)/nm: 206 (log  4.14), 275 (log  3.12); IR (ATR) νmax 3361 (br), 2929, 1702, 1609, 1457, 1407 cm-1; 1H NMR and 13C NMR (Table 5); LRESIMS m/z 307.6 - + + (M-H) , m/z 309.5 (M+H) ; HRESIMS calcd. for C18H28O4Na (M+Na) m/z 331.1880, found 331.1886.

Marilone G (7): white amorphous solid (208 µg/L, 0.042 %); UV λmax (MeOH)/nm: -1 1 220 (log  4.17), 266 (log  3.73); IR (ATR) νmax 3268 (br), 2930, 1697, 1605 cm ; H

NMR (MeOD): 1-H: δ 5.54, dd (1.6, 6.5); 6-H: δ 6.87, s; 9-Ha: 3.08, dd (6.5, 19.1); 9- 13 Hb: 2.50, dd (1.6, 19.1); 10-H3: 4.01, s; 11-H3: 2.23, s; C NMR (MeOD): CH-1, δ 67.1; qC-2, δ 139.4; qC-3, δ 158.4; qC-4, δ 128.1; qC-5, δ 159.8; qC-6, δ 103.3; qC-7, δ

137.2; qC-8, δ 206.4; CH2-9, δ 48.4; CH3-10, δ 61.1; CH3-11, δ 10.0; (2D NMR in Appendix – Complete 2D NMR data); LRESIMS m/z 206.9 (M-H)-; HRESIMS calcd. - for C11H11O4 (M-H) m/z 207.0652, found 207.0650.

Marilone H, 5-ethyl-3-hydroxy-2-methyl-pyran-1-one (8): amorphous solid (0.483 ug/L, 0.098 %); UV λmax (MeOH)/nm: 207 (log  4.05), 285 (log  3.69); IR (ATR) νmax -1 1 3330 (br), 2929, 2674, 1645, 1578 cm ; H NMR (acetone-d6): 4-H: δ 6.02, s; 6-H2: δ 13 2.42, q (7.5); 7-H3: δ 1.13, t (7.5); 8-H3: δ 1.81, s; C NMR (acetone-d6): qC-1, δ 166.0; qC-2, δ 98.2; qC-3, δ 165.6; CH-4, δ 99.1; qC-5, 164.9; CH2-6, δ 27.1; CH3-7, δ 11.3;

CH3-8, δ 8.5; (2D NMR in Appendix – Complete 2D NMR data); LRESIMS m/z 153.1 - + (M-H) , HREIMS calcd. for C8H10O3 (M) m/z 154.0630, found 154.0628.

Marilone I (10): white amorphous solid (160 µg/L, 0.084 %); UV λmax (MeOH)/nm: -1 1 214 (log  4.09), 272 (log  2.98) IR (ATR) νmax 2930, 1610, 1463 cm ; H NMR and 13 + C NMR (Tables 1 and 2); LREIMS m/z 330.2 (M) ; HREIMS calcd. for C23H34O5 + (M) m/z 390.2406, found 390.2417.

Marilone J (11): white amorphous solid (170 µg/L, 0.089 %); UV λmax (MeOH)/nm: -1 1 214 (log  4.10), 272 (log  2.98); IR (ATR) νmax 2925, 1610, 1464 cm ; H NMR and 13 + C NMR (Tables 1 and 2); HREIMS calcd. for C23H34O5 (M) m/z 390.2406, found 390.2412.

Methodology for the performed biological assays. As described in chapter 3 - General methodology.

6. Stachylines A – D from the Sponge-derived Fungus Stachylidium sp.

Abstract The marine-derived fungus Stachylidium sp. was isolated from the sponge Callyspongia cf. C. flammea. Four new, putatively tyrosine-derived and O-prenylated natural products, stachylines A – D (1 – 4), were obtained from the fungal extract. The structures of 1 – 4 were elucidated based on extensive spectroscopic analyses. The absolute configuration of compound 2 was established by Mosher’s method. Stachyline A (1) possesses a rare terminal oxime group and occurs as an interchangeable mixture of E/Z- isomers.

4' E- HO 5 5' N 2' 1' OH O OH O 7 1 3 O OH

N Z- HO O OH

(1) (2)

OH OH O O OH O OH

(4) (3) Figure 1. Structure formulae of Compounds 1 - 4

Introduction

The marine environment harbours approximately half of the global biodiversity and is estimated to contain between 3 and 500 million different species, offering an almost infinite resource for novel compounds (Tziveleka et al., 2003). Among these organisms marine-derived fungi became known as prolific producers of structurally most intriguing compounds (Bhadury et al., 2006). In general, tyrosine derivatives have only rarely been reported from fungi, and in most cases such compounds were obtained from strains originating from environmentally extreme habitats, e.g. tyrosol carbamate which was isolated from the deep-water fungus Arthrinium sp. (Sashidhara et al., 2009). Phytomyces sp., producing O-prenylated tyrosine derivatives, is an extremophile collected from an acid mine waste rich in toxic metals (Stierle et al., 2003). Another unusual case is aspergillusol A, an α-glucosidase inhibitor obtained from the sponge- derived fungus Aspergillus aculeatus which is reported to be the only known fungal tyrosine derivative to possess an oxime group (Ingavat et al., 2009). Secondary metabolites with an oxime substituent are rare, and most of the reported examples have potent bioactivity, e.g. the actinomycete-derived nocardicins displayed strong antibiotic activity (Hashimoto et al., 1976), and brevioxime from Penicillium brevicompactum inhibited the biosynthesis of insect juvenile hormones (Amade et al., 1993). P. olsonii produced 2-(4-hydroxyphenyl)-2-oxoacetaldehyde oxime (PHBA) which regenerated phosphorylated cholinesterase (Moya et al., 1997). The oxime geometrical isomers collismycins A and B were isolated from Streptomyces sp. MQ22 which inhibited dexamethasone glucocorticoid receptor binding (Shindo et al., 1994).

During our search for new cytotoxic natural products an extract of the marine-derived fungus Stachylidium sp. was found to be active. During chromatographic separations it became clear that this fungus produces a vast array of secondary metabolites with intriguing structural features, among them the four novel, putatively tyrosine-derived and O-prenylated natural products, stachylines A – D (1 - 4). Stachyline A (1) is distinguished by an oxime terminal group, probably derived through biosynthetic reactions similar to those known for cyanogenic glycosides and nocardicin A formation

(Cimino et al., 1975; Isaaki et al., 1993; Bjarnholt and MØller, 2008; Kelly and Townsend, 2002; Kelly and Townsend, 2002). The molecules were evaluated in a number of biological assays, to date however no considerable activity was detected.

Results

The RP-18 HPLC chromatogram of 1 contained two peaks (ratio 1:1), which when re- injected after their individual isolation, again resulted in the same chromatogram. This result suggested that compound 1 exists as a mixture of interchangeable isomers. NMR spectra also presented two sets of data, one for each isomer (see Tables 1 and 2). For clarity in the description of the structure elucidation, only one set of data will be considered initially. The molecular formula of 1 was deduced by accurate mass measurement 13 (HREIMS/HRESIMS) to be C13H17NO3, requiring six sites of unsaturation. The C NMR and DEPT135 spectra showed the presence of thirteen resonances for one methyl, five sp2 methine, one sp3 methine, one sp2 methylene, two sp3 methylene groups and three quaternary carbons (see Tables 1 and 2). The UV maximum at 276 nm indicated the presence of an aromatic moiety, whereas an IR absorption at around 3300 cm-1 arose from a hydroxy group in the molecule. 1H NMR and 1H-13C HSQC data (see Tables 1 and 2) included two resonances for magnetically equivalent protons, i.e. H-2/H-6 and H-3/H-5 resonating at δH 7.17 and 6.88, respectively. This spectroscopic feature was interpreted as characteristic for a para-substituted aromatic ring. From the same spectra the presence of an exomethylene 13 moiety, CH2-4’, was evident (δH 4.89, 5.08; δC 112.1). The C NMR spectrum showed two resonances for carbons connected to oxygen atoms, namely at δC 72.1 (C-1’) and

73.9 (C-2’) (see Table 1), and the protons attached to these carbon atoms, H2-1’ and H- 2’ were coupled as evidenced by 1H-1H COSY correlations. 1H-1H COSY correlations indicated the presence of another spin system between H2-4’ and H3-5’. The partial structures deduced from these two spin systems were connected making use of 1H-13C

HMBC correlations. Thus, correlations from H3-5’ to C-2’, C-3’ and C-4’ delineated an unsaturated and hydroxylated isoprene unit (see Appendix – Complete 2D NMR data).

This was confirmed by further heteronuclear long range correlations from H2-4’ to C-3’ and C-2’. 1H-13C HMBC correlations between H-1’ to qC-4 showed that the isoprene unit was connected via C-4 to the para-substituted aromatic moiety. Due to the downfield shift of C-1’ and C-4 (δC 72.1, 158.5) connection of both parts of the molecule occurred through an oxygen atom.

According to the molecular formula, the second substituent on the aromatic ring of 1 1 13 must have the composition C2H4NO. H- C HMBC correlations could be detected from the aromatic protons H-2/H-6 to the methylene carbon C-7, making the connection of the second side chain via C-1 most likely. Through an 1H-1H COSY experiment, the methylene protons CH2-7 were shown to be part of a spin system including H-8 (δH 6.73), whereby the latter was bound to an sp2 hybridized carbon. The remaining presence of a nitrogen, oxygen and proton atom was assigned to an unusual oxime moiety (see Tables 1 and 2). The latter is known to be able to adopt interchangeable configurations (Tennant et al., 1979). E- and Z- isomers in the case of 1 presented clearly distinguishable NMR shifts for the C-8 to C-2/C-6 part of the molecules (see Tables 1 and 2). The assignment of NMR shifts for the E- and Z- isomers was supported using the ACD NMR predictor software (ACD/Labs®). The configuration at C-2’ of compound 2 was found to be S as deduced by Mosher’s method. Based on the negative optical rotation of compound 1, which was also observed for 2, and the close biosynthetic relationship between both compounds, the S configuration is also proposed for C-2’ of 1 (see Figure 2). For compound 1 the trivial name E- and Z-stachyline A is proposed. The molecular formula of 2 was deduced by accurate mass measurement (HREIMS) to 13 1 be C13H16O4, requiring six sites of unsaturation. The C and H NMR spectra were closely related to those of compound 1 (see Table 2). One of the substituents on the aromatic ring was again an O-prenyl moiety as in 1, also connected via qC-4 and C-1’ (see Tables 1, 2 and Appendix – complete 2D NMR data). The second substituent on the aromatic ring of 2 was however, different and established by interpretation of 1H-13C

HMBC long range correlations. Thus, the methylene protons H2-7 had a heteronuclear coupling with C-2 and C-6 of the aromatic ring. Additionally, H2-7 had HMBC long range correlations to the quaternary carbon C-8, which according to its characteristic 13 C NMR resonance was part of a carboxylic acid group (δC 173.0). Compound 2 was thus an O-prenylated phenyl acetic acid. The absolute configuration at C-2’ was determined by modified Mosher’s method. The evaluation of the corresponding 1H-13C HSQC NMR spectra revealed differences of the proton shifts between the (S)- and (R)-MTPA esters (see Figure 2 and Appendix – complete 2D NMR data). The calculated differences in chemical shifts [δ of protons in the (S)-MTPA-ester minus δ of the corresponding protons in the (R)-MTPA-ester] led to the assignment of the (S)-absolute configuration for C-2´. Since compounds 1 - 4 all

showed a negative specific optical rotation, it was assumed that they all share the (S-) configuration at C-2’. Compound 2 is an O-prenylated phenyl acetic acid, for which we propose the trivial name stachyline B.

Figure 2. Deduction of the absolute configuration at C-2´ using modified Mosher’s method with ΔS-R values of MTPA-esters of compound 2.

The molecular formula of 3 was deduced by accurate mass measurement (HREIMS) to be C13H18O3, requiring five sites of unsaturation. 1D and 2D NMR data of 3 were closely similar to those of 2 (see Tables 1, 2 and Appendix – Complete 2D NMR data), except for the absence of resonances for the carboxylic acid group in the 13C NMR spectrum and the presence of an additional resonance for a sp3 methylene group. The 13 latter had a downfield shifted C NMR resonance (δC 64.1), which evidenced connection to an oxygen atom. Thus, 3 can be regarded as the C-8 reduction product of 2, for which the trivial name stachyline C is suggested. The molecular formula of 4 was deduced by accurate mass measurement (HRESIMS) to be C13H18O4, requiring five sites of unsaturation. 1D and 2D NMR spectroscopic data of compound 4 exhibited many identical features when compared with those of 2 (see Tables 1, 2 and Appendix – complete 2D NMR data). A difference, however concerned the exo-methylene proton signals which were absent in the 1H NMR spectrum of 4, whilst signals for a methyl (H3-4’, δH 0.97) and a methine group were present (H-3’, δH 1.86). 1H-1H COSY correlations confirmed the structure, since a spin system connected all atoms within the isoprene unit from H2-1’ to H3-5’ and H3-4’ (see Table 2 and Appendix – complete 2D NMR data). For compound 4 the trivial name stachyline D is proposed.

Table 1: 13C NMR Spectroscopic Data for Compounds 1 - 4.

1* 2 3 4 a, b, c a, b, c a, b,c a, b, c position δC, mult. δC, mult. δC, mult. δC, mult. 1 130.1, qC 128.0, qC 132.5, qC 127.9, qC 2 130.5, CH 131.2, CH 130.7, CH 131.2, CH 3 115.5, CH 115.3, CH 115.2, CH 115.2, CH 4 (Z-), 158.5, qC (E-), 158.7, qC 158.8, qC 158.3, qC 159.0, qC 5 115.5, CH 115.3, CH 115.2, CH 115.2, CH 6 130.5, CH 131.2, CH 130.7, CH 131.2, CH

7 (Z-), 31.0, CH2 (E-), 35.4, CH2 40.5, CH2 39.4, CH2 40.4, CH2

8 (Z-), 150.0, CH (E-), 149.7, CH 173.0, qC 64.1, CH2 173.2, qC

1’ 72.1, CH2 72.1, CH2 72.1, CH2 71.6, CH2 2’ 73.9, CH 73.9, CH 74.0, CH 74.6, CH 3’ 146.1, qC 146.1, qC 146.2, qC 31.6, CH

4’ 112.1, CH2 112.1, CH2 112.0, CH2 17.7, CH3

5’ 18.7, CH3 18.8, CH3 18.8, CH3 19.4, CH3 a b acetone-d6,, 75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY, see Appendix – 2D NMR complete data). c Implied multiplicities determined by DEPT. *Carbon resonances were attributed to the (Z-) or (E-) configuration, according to ACD/NMR Predictor® software.

Table 2. 1H NMR Spectroscopic Data for Compounds 1 - 4.

1* 2 3 4 a, b a, b a, b a, b position δH (J in Hz) δH (J in Hz) δH (J in Hz) δH (J in Hz) 1 2 (Z-), 7.17, d (8.4) (E-), 7.13, d (8.4) 7.20, d (8.4) 7.13, d (8.4) 7.19, d (8.4) 3 6.88, d (8.4) 6.87, d (8.4) 6.84, d (8.4) 6.87, d (8.4) 4 5 6.88, d (8.4) 6.87, d (8.4) 6.84, d (8.4) 6.87, d (8.4) 6 (Z-), 7.17, d (8.4) (E-), 7.13, d (8.4) 7.20, d (8.4) 7.13, d (8.4) 7.19, d (8.4) 7 (Z-), 3.60, d (5.5) (E-), 3.39, d (6.2) 3.51, br s 2.72, t (7.1) 3.52, br s 8 (Z-), 6.73, t (5.5) (E-), 7.40, t (6.2) 3.68, t (7.1) 1’ a: 4.01, dd (3.7, 9.5) a: 4.01, dd (4.0, 9.8) a: 4.00, dd (4.3, 9.8) a: 4.00, dd (4.0, 9.8) b: 3.90, dd (7.3, 9.5) b: 3.90, dd (7.2, 9.8) b: 3.89, dd (7.2, 9.8) b: 3.89, dd (6.6, 9.8) 2’ 4.39, m 4.40, dd (4.0, 7.2) 4.39, dd (4.3, 7.2) 3.68, m 3’ 1.86, m 4’ a: 4.89, br s a: 4.88, br s a: 4.89, br s 0.97, d (6.8) b: 5.08, br s b: 5.08, br s b: 5.09, br s 5’ 1.78, s 1.79, s 1.79, s 0.97, d (6.8) a b acetone-d6, 300 MHz. Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY see Appendix – 2D NMR complete data). *Proton resonances were attributed to the (Z-) or (E-) configuration, according to ACD/NMR Predictor® software.

Further compounds isolated were established to be known molecules, including etrogol (5) (Stierle et al., 2003), tyrosol (6) (Hashimoto et al., 1976), phenyl acetic acid (7) (Nair and Burke, 1988), benzoic acid (8) (Chen, 1958) and para-hydroxyphenyl acetic acid (9) (Chen, 1958; Crowden and Ralph, 1961).

Figure 3. Structural formulae of the known compounds 5 - 9

Biological activity

Stachyline A, B and C (1, 2, 3) were evaluated for cytotoxic activity against a panel of 3 cancer cell lines (NCI-H460, MCF7 and SF268, tested at the 100 µM dose), but did not show activity. Stachyline A and B (2) were tested each against a panel of forty four psychoactive receptors, namely 5ht1a, 5ht1b, 5ht1d, 5ht1e, 5ht2a, 5ht2b, 5ht2c, 5ht3, 5ht5a, 5ht6, 5ht7, Alpha1A, Alpha1B, Alpha1D, Alpha2A, Alpha2B, Alpha2C, Beta1, Beta2, Beta3, BZP Rat Brain Site, D1, D2, D3, D4, D5, DAT, DOR, Gaba A, H1, H2, H3, H4, KOR, M1, M2, M3, M4, M5, MOR, NET, SERT, Sigma1, Sigma2, (activity considered with at least 50 % inhibition at the 10 μM level), but exhibited no potent activity. One could mention though the 48.3 % antagonistic effect of Stachyline B specifically against the Adrenergic receptor Alpha 2C (at 10 µM dose, quadruplicate measurements). Stachylines C (3) and D (4) were also tested against five dopamine receptors (D1 – D5) but also did not show activity. Stachyline A (1) was tested in antibacterial (Escherichia coli, Bacillus megaterium), antifungal (Mycotypha microspora, Eurotium rubrum, and Microbotryum violaceum), antialgal (Chlorella fusca) assays at a dose of 50 µg/ml disc level but did not show activity. Stachyline B (2) was further tested in protein kinases DYRK1A and CDK5 inhibition activity assay (tested at 10 mM dose), for antiplasmodial activity assay against Plasmodium berghei (tested at 25 µM single dose), against two antibiotic resistant Mycobacterium tuberculosis strains, MTB72 and R46 (tested at the 50 µg/ml disc level), for the inhibition of the NF-kB protein complex assay (at the 100 µM) and in a panel of anti-diabetic activity assays: PPAR ligand assay, PPAR co-activator assay, PTP-1B inhibitor assay and glucose uptake assay (both 3T3-l1 cells and primary rat/mouse adipocytes, tested at the 100 µM dose) but did not show activity in any of the tests. Stachyline D (3) was evaluated against Influenza B virus (tested at 100 µg/mL dose) and for the inhibition of the viral HIV-1 and HIV-2 induced cytopathogenic effect in MT-4 cells (tested at the 50 µg/mL dose) but did not show activity.

Discussion

Stachylines A-D are presumably tyrosine-derived metabolites, a group of compounds rarely encountered in fungal metabolism. The oxime group in compound 1 is even more intriguing. Phenylpyruvic acid oxime derivatives (Isaaki et al., 1993), brominated tyrosine-derived compounds like the bastadins and psammaplins (Calcul et al., 2010), as well as biosynthetically possibly related nitrile containing metabolites (Flemming, 1999) occur in marine sponges. The Stachylidium sp. used for this investigation was isolated from a marine sponge. It was observed that the configuration of the oxime moiety in compound 1 was labile, and upon chromatographic isolation of the E- and Z-isomers, respective isomerisation took place instantaneously. None of the isomers seemed to be thermodynamically more stable under the prevailing conditions, since a 1:1 ratio of isomers was obtained (see Appendix – Spectroscopic data supporting information). In contrast to that, the geometrical isomers collismycin A and B from Streptomyces sp. MQ22 were reported to be not interchangeable in many organic solvents and at room temperature. Only by heating in ortho-dichlorobenzene at 120 °C they were gradually interchangeable (Shindo et al., 1994). For 2-(4-hydroxyphenyl)-2-oxoacetaldehyde oxime (PHBA) isolated from Penicillium olsonii, the E-configuration was described without mentioning any isomerisation reactions (Amade et al., 1993). Although the oxime functional groups are rare in natural products they occur in a variety of phyla, e.g., sponges, bacteria, fungi, and plants. Tyrosine-derivatives with an oxime moiety were found in actinomycetes, such as the β-lactam antibiotic nocardicin A from Nocardia uniformis (Hashimoto et al., 1976). Higher plants are also capable of producing oxime groups, since the biosynthesis of glucosinolates and cyanogenic glycosides, e.g. dhurrin from Sorghum bicolor proceeds via aldoximes (Bjarnholt and

MØller, 2008; Flemming, 1999; Akazawa et al., 1960; Sibbesen et al., 1995). From these studies, oximes can be regarded as biosynthetic precursors of nitriles. Aspergillusol A was reported to be the first fungal tyrosine-derivative with an oxime moiety (Ingavat et al., 2009), however a previous publication had described the isolation of 2-(4-hydroxyphenyl)-2-oxoacetaldehyde oxime (PHBA) from Penicillium olsonii (Amade et al., 1993). Hence, the current report is the third one of a fungal tyrosine-derived oxime derivative.

Concerning the biosynthesis of naturally occurring oximes it is not clear which isomer is produced initially. The aldoxime intermediate in the biosynthesis of the cyanogenic glycoside dhurrin has been reported to initially have the E-configuration, subsequently undergoing non-enzymatic isomerisation (Bjarnholt and MØller, 2008; Sibbesen et al. 1995). Nocardicin A with Z-oxime configuration is thought to be preferentially produced over the E-configurated nocardicin B, due to favourable intramolecular hydrogen-bonding in biosynthetic intermediates (Kelly and Townsend, 2002; Kelly and Townsend, 2005). In the case of bastadins and psammaplins, it was assumed that the Z- oxime configuration is initially produced during biosynthesis, followed by isomerisation to the thermodynamically more favored E-isomers during extraction and isolation of the compounds (Calcul et al., 2010). Stachylines A-D are proposed to be biosynthetically derived from tyrosine (Figure 4). For stachyline A the biosynthetic process may be similar to that of cyanogenic glycosides, e.g. dhurrin, in that the precursor tyrosine is N-hydroxylated by CYP 450 enzymes to N,N-dihydroxytyrosine and subsequently decarboxylated and dehydrated to form an aldoxime (Bjarnholt and MØller, 2008). A similar pathway was described for the norcardicin biosynthesis (Kelly and Townsend, 2002; Kelly and Townsend, 2005).

NH2 HOOC OH

oxidative desamination N-hydroxylation decarboxylation

HO N OH HOOC HOOC OH OH

oxidative decarboxylation dehydration

HO N OH

(E)-p-hydroxyphenyl acetaldoxime

prenylation

PPO

hydroxylation

HO HOOC N O OH O OH

Stachyline B

reduction hydrogenation non-enzymatic isomerisation

HO HOOC HO N O OH O OH O OH

Stachyline C Stachyline D Stachyline A (E-, Z-)

Figure 4. Proposed biosynthesis of stachylines A – D.

Experimental section General Experimental Procedures. See chapter 3 - General methodology.

Fungal material. See chapter 3 - General methodology.

Culture, extraction and isolation. Compounds 1 to 4 were isolated from three different cultures of Stachylidium sp. The first and second culture comprised 12 L and 10 L of agar-biomalt medium (biomalt 20 g/L, 15 g/L agar) supplemented with sea salt incubated for 2 months (12 L) and 40 days (10 L) in Fernbach flasks at room temperature. The third culture was performed with 9.6 L liquid YPM medium (yeast extract 5 g/L, peptone from YPM 3 g/L, mannitol 25 g/L) incubated for 10 days. An extraction with 5 L EtOAc yielded 5.9 g, 2.1 g and 1.0 g of extract, respectively which were subjected to a VLC fractionation on silica gel, using a step gradient solvent system with petroleum ether - acetone from 10:1, 5:1, 2:1, 1:1 to 100 % acetone and, subsequently 100 % MeOH, resulting in 6 VLC fractions for each culture. Compounds 2 and 3 were isolated from the first culture (12 L), whilst compound 4 was isolated from the second culture (10 L) and compound 1 was isolated from the third culture (YPM medium). The known compounds 6 and 9 were isolated from the first culture and the know compounds 5, 7 and 8 from the first and third culture (isolation described for the YPM culture). Compound 1 was isolated from VLC fraction 3 by NP-HPLC separation using petroleum ether - acetone (7:1) to yield 6 fractions. Sub-fraction 3 and 4 corresponded to a mixture of isomers of compound 1 (E- and Z-, 4.4 mg), which was again separated by RP-HPLC using 50 % MeOH (tR 11 min and 15 min), but immediately formed the initial mixture of isomers in a ratio of 1:1. Compounds 2 and 3 were isolated from VLC fraction 3 by NP-HPLC using petroleum ether - acetone (11:2) to yield 10 fractions. Further RP-HPLC fractionation with sub-fraction 5 using 50 % MeOH yielded 5 more sub-fractions, in which sub-fraction 4 was a mixture of compounds 2 and 3. Finally, we performed NP-HPLC fractionation using petroleum ether - acetone (7:1) to afford compound 2 (fraction 1 of 2, 4.1 mg, tR 19 min) and compound 3 (fraction 2 of 2, 2.2 mg, tR 26 min). Compound 4 was isolated from VLC fraction 4, followed by HPLC fractionation using petroleum ether - acetone (9:2; sub-fraction 2 of 7) which was further purified using RP-HPLC with 60 % MeOH (fraction 2 of 4, 1.8 mg, tR 10 min). Etrogol (5) was isolated from VLC fraction 3, followed by HPLC fractionation using petroleum ether - acetone (7:1) to yield 5 sub-fractions. Sub fraction 1 was further purified using petroleum ether - acetone (7:1) to yield pure etrogol (fraction 3 of 3, 3.1 mg, tR 26 min). Tyrosol (6) was isolated from VLC fraction 3 followed by HPLC fractionation using petroleum ether - acetone (5:1) to yield 10 fractions. Further RP- HPLC fractionation with sub-fraction 6 using 30 % MeOH yielded the pure compound

(fraction 3 of 7, 2.5 mg, tR 19 min). Phenyl acetic acid (7) and benzoic acid (8) were isolated from VLC fraction 3, followed by HPLC fractionation using petroleum ether - acetone (11:1) to yield 5 fractions. Benzoic acid was present in fraction 2 (7.9 mg, tR 16 min) and phenylacetic acid in fraction 3 (11.2 mg, tR 25 min). Para-hydroxyphenyl acetic acid (9) was isolated from VLC fraction 3 followed by HPLC fractionation using petroleum ether - acetone (5:1) to yield 10 fractions. Further RP-HPLC fractionation with sub-fraction 6 using 30 % MeOH yielded the pure compound (fraction 4 of 7, 1.8 mg, tR 27 min).

Stachyline A, mixture of E-/Z-isomers, 1:1 (1): white amorphous solid (458 μg/L, 23 0.43 %); [α] D -15 (c 0.29, MeOH); UV (MeOH) λmax (log ε) 225 nm (3.60), 222 nm -1 1 (2.84), 276 nm (2.76) ; IR (ATR) νmax 3300 (br), 2922, 1610, 1510 cm ; H NMR and 13C NMR (Tables 1 and 2 and Appendix – Complete 2D NMR data); ESIMS m/z 236.1 + + [M+H] ; HREIMS m/z 235.1208 [M] (calcd. for C13H17NO3, m/z 235.1208); + HRESIMS m/z 258.1125 [M+Na] (calcd. for C13H17NNaO3, m/z 258.1101).

23 Stachyline B (2): white amorphous solid (340 μg/L, 0.07 %); [α] D - 12 (c 0.33,

MeOH); UV (MeOH) λmax (log ε) 204 nm (3.35), 227 nm (3.75), 276 nm (3.05), 283 nm –6 (2.75); CD (c 1.06 × 10 mol/L, MeOH), λ (ε) = 197 (+ 1.78); IR (ATR) νmax 3364 (br), 2929, 1610, 1511 cm-1; 1H NMR and 13C NMR (Tables 1 and 2 and Appendix – Complete 2D NMR data); EIMS m/z 236.1 [M]+; HREIMS m/z 236.1051 [M]+ (calcd. for C13H16O4, m/z 236.1049).

23 Stachyline C (3): white amorphous solid (183 μg/L, 0.04 %); [α] D - 16 (c 0.33,

MeOH); UV (MeOH) λmax (log ε) 205 nm (3.43), 225 nm (3.85), 277 nm (2.95), 283 nm –6 (2.95); CD (c 1.25 × 10 mol/L, MeOH) λ (ε) = 199 (+ 1.94); IR (ATR) νmax 3235 (br), 2937, 2551, 1685, 1509 cm-1; 1H NMR and 13C NMR (Tables 1 and 2 and Appendix – Complete 2D NMR data); EIMS m/z 222.1 [M]+; HRESIMS m/z 245.1162 + [M+Na] (calcd. for C13H18NaO3, m/z 245.1148).

23 Stachyline D (4): white amorphous solid (180 µg/L 0.09 %); [α] D - 6 (c 0.12, MeOH);

UV (MeOH) λmax (log ε) 205 nm (3.86), 226 nm (3.86), 276 nm (3.08), 283 nm (2.90); -1 1 13 IR (ATR) νmax 3265 (br), 1687, 1512 cm ; H NMR and C NMR (Tables 1 and 2 and

Appendix – Complete 2D NMR data); ESIMS m/z 237.1 [M-H]-; HRESIMS m/z + 261.1096 [M+Na] (calcd. for C13H18NaO4, m/z 261.1097).

Preparation of the (R)- and (S)-MTPA esters of compound 2. The secondary alcohol (1 eq, 1 mg, 4.23 x 10-6 µmol) was dissolved with the corresponding MTPA-Cl (10 eq) in an adequate quantity of deuterated pyridine and DMAP (1 eq) in an NMR tube. The reaction was followed by 1H-NMR to observe the downfield proton shift of H-2’. After 1 hour reaction time, the final 1H-NMR was recorded, the sample dried and re-dissolved in D-acetone for 1H-13C HSQC measurements. The Δ(S-R) values between (S)- and (R)- MTPA esters were recorded with the help of both 1H-NMR and 1H-13C HSQC (see Figure 1 and Appendix - Spectroscopic data supporting information).

Bioassays. See chapter 3 - General methodology.

7. Novel Furyl Derivative from the Sponge-derived Fungus Stachylidium sp.

Abstract The marine-derived fungus Stachylidium sp. was isolated from the sponge Callyspongia sp. cf. C. flammea. One new furyl derivative, 2-(furan-3-yl)acetic acid (1) were isolated from the fungal extract which was obtained from a agar-BMS medium supplemented with artificial sea salt. The structure of 1 was elucidated based on extensive spectroscopic analysis. Three further and known furyl derivatives, namely 1-(furan-2- yl)ethane-1,2-diol (2), furan-2,5-diyldimethanol (3) and 2-furoic acid (4) were found.

O O OH 1 5 COOH 3 OH (1) (2)

O O HO OH CH2OH

(3) (4) Figure 1. Structures of Compounds 1 - 4

Introduction

Interactions that occur between organisms are often mediated by small molecules, e.g. N-acyl-homoserine lactones, 2-heptyl-3-hydroxy-4-quinoline (PQS) (Dudler and Eberl, 2006; Camili and Bassler, 2006). In addition to controlling the production of active compounds in bacteria, there is a growing number of reports indicating that small molecules like N-acyl-homoserine lactones autoinducers modulate gene expression of eukaryotic organisms (Sperantio, 2004; Shinner et al., 2005). Furyl derivatives are also found in a wide taxonomical range of bacterial and fungal species. Methyl 2-furancarboxylate is found in Loktanella (Dickschat et al., 2005), while the related compound 2-acetylfuran (Andreae, 1990) is a metabolite of myxobacteria (Dickschat et al., 2004; Dickschat et al., 2005a), Streptomyces citreus (Pollak and Berger, 1996) and marine bacteria (Dickschat et al., 2005b). Interestingly, the typical fungal metabolite 3-methylfuran has been reported from three Streptomyces strains (Schöller et al., 2002; Schulz and Dickschat, 2007), whereas the regioisomer 2- methylfuran is reported from Thermoactinomyces (Wilkins, 1996). During our search for new natural products produced from the marine-derived fungus Stachylidium sp., one new (1) and three known furyl derivatives (2 – 4) were isolated from a culture of this fungus on agar-BMS media supplemented with artificial sea salt.

Structure elucidation

The molecular formula of 1 was established by HREIMS to be C6H6O3, requiring four sites of unsaturation. The 13C NMR and DEPT135 spectra indicated the presence of six resonances, including three sp2 methines, one sp3 methylene and two quaternary carbons (Compound Spectroscopic Description and Table 1). The three methine protons were assigned to be part of a furyl moiety. H-1 and H-4 have downfield shifted proton resonances in the 1H-NMR spectra, indicating that the furyl oxygen atom is placed between them. H-2 was placed next to H-1 making use of 1H-1H-COSY correlations. 3 Key heteronuclear long range correlations from the sp CH2-5 to the furyl carbons C-2,

C-3, C-4 and the carboxyl carbon C-6 (δc 172.3) completed the assignment of 1 as 2- (furan-3-yl)acetic acid. Compound 1 is a new natural products but was previously synthesized (Paolucci and Rosini, 2007; Kassianidis et al., 2005).

The structure of the furyl derivatives 2, 3 and 4 was established to be namely 1-(furan- 2-yl)ethane-1,2-diol (2) , furan-2,5-diyldimethanol (3) and 2-furoic acid (4), respectively by comparison of spectroscopic data with those previously reported for these natural products (Lu et al., 2008, Schneider et al., 1996; Hong et al., 1981).

Table 1. 1D and 2D NMR spectroscopic data for compound 1

a, b, e a, b a, c a, d position δC, mult. δH (J in Hz) COSY HMBC 1 143.6, CH 7.476, m 2 2, 3, 4 2 112.5, CH 6.43, s (br) 1 1, 3, 4, 5 3 119.1, qC 4 141.3, CH 7.482, m 5 1, 2, 3, 5

5 30.6, CH2 3.45, s 4 2, 3, 4, 6 6 172.3, qC a b CD3COCD3, 300/75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT.

(1) Figure 2. Key HMBC correlations of compound 1

Biological activity assays

(R)-1-(furan-2-yl)ethane-1,2-diol (2) were tested for antiplasmodial activity assay against Plasmodium berghei (tested at 25 µM single dose), in antibacterial (Escherichia coli, Bacillus megaterium), antifungal (Mycotypha microspora, Eurotium rubrum, and Microbotryum violaceum), antialgal (Chlorella fusca) assays at a dose of 50 µg/ml disc level but did not show any activity.

Compounds 1 – 4 are currently being tested for agonistic/antagonistic quorum sensing effect in the bacterial species Chromobacterium violaceum.

Discussion

Small molecules like furyl derivatives are widely present in fungal and bacterial extracts (Dickschat et al., 2004; Dickschat et al., 2005a; Dickschat et al., 2005b; Pollak and Berger, 1996; Schöller et al., 2002; Schulz and Dickschat, 2007), and they could work in the quorum sensing responsible for eukaryotic-bacterial as well as bacterial-bacterial chemical interactions, (Dudler and Eberl, 2006). Interestingly, peptides, phthalides and phthalimidines indicating the presence of bacterial metabolism were isolated from the same fungal extract (unpublished data). We are currently undergoing studies to identify the possible origin of these compounds, as well as the possible interaction/origin of the hereby reported furyl derivatives.

Experimental section General Experimental Procedures. See chapter 3 - General methodology.

Fungal material. See chapter 3 - General methodology.

Culture, extraction and isolation. Compounds 1 and 2 were isolated from two different cultures of Stachylidium sp., the first and second performed on 12 L and 10 L respectively of agar-biomalt medium (biomalt 20g/L, 15 g/L agar) supplemented with sea salt during 2 months (12 L) and 40 days (10 L). An extraction with 5 L EtOAc yielded respectively 5.9 g and 2.1 g of extract which were subjected to a VLC fractionation in a silica open column using a gradient solvent system with petroleum ether - acetone at 10:1, 5:1, 2:1, 1:1, 100% acetone and 100% MeOH, resulting thus 6 VLC fractions from each culture. Compound 2, 3 and 4 were isolated from the first culture (12 L), whilst compound 1 was isolated from the second culture (10 L). Compound 1 was isolated from VLC fraction 3, followed by HPLC fractionation using petroleum ether - acetone 5:1 (sub-fraction 2 of 10) which was further purified using

NP-HPLC with petroleum ether - acetone 9:1 (fraction 3 of 4, 3.2 mg, tR 21 min).

Compound 2 was isolated from VLC fraction 3, followed by HPLC fractionation using petroleum ether - acetone 5:1 (sub-fraction 7 of 10) which was further purified by HPLC reverse phase fractionation using 50% MeOH (sub-fraction 1 of 4). Finally we used RP-HPLC using 10% MeOH to yield the pure compound (fraction 2 of 7, 3.1 mg, tR 4 min). Compound 3 was isolated from VLC fraction 3, followed by HPLC fractionation using petroleum ether - acetone 5:1 (sub-fraction 7 of 10) which was further purified by HPLC reverse phase fractionation using 50% MeOH (sub-fraction 1 of 4). Finally we used RP-HPLC using 10% MeOH to yield the pure compound (fraction 3 of 7, 2.6 mg, tR 4 min). Compound 4 was isolated from VLC fraction 3, followed by HPLC fractionation using petroleum ether - acetone 5:1 (sub-fraction 7 of 10) which was further purified by HPLC reverse phase fractionation using 50% MeOH (sub-fraction 1 of 4). Finally we used RP-HPLC using 10% MeOH to yield the pure compound (fraction 4 of 7, 1.6 mg, tR 4 min).

2-(furan-3-yl)acetic acid (1): white amorphous solid (320 µg/L 0.152 %); UV (MeOH) -1 1 λmax 206 nm (log Є 3.76); IR (ATR) νmax 2907 (Br), 1693 cm ; H NMR [H-1, δ 7.476, m; H-2, δ 6.43, s (br); H-4, δ 7.482, m; H-5, δ 3.45, s] and 13C NMR (C-1, δ 143.6, CH;

C-2, δ 112.5, CH; C-3, δ 119.1, qC; C-4, δ 141.3, CH; C-5, δ 30.6, CH2; C-6, δ 172.3, qC; 2D NMR in Table 1); LRESIMS m/z 124.9 (M-H)-; HRESIMS calcd. m/z 125.0233 - for C6H5O3 (M-H) , found 125.0240.

Methodology for the performed biological assays. See chapter 3 - General methodology.

8. Endolides A – J, N-methylated Peptides from the Sponge-derived Fungus Stachylidium sp.

Abstract Marine organisms are rich sources of structurally diverse bioactive compounds. Recently, a great deal of interest has been expressed regarding marine-derived bioactive peptides because of their numerous health beneficial effects (Kim and Wijesekara, 2010). During our search for new natural products produced from the marine-derived fungus Stachylidium sp., ten novel cyclic N-methylated peptides, Endolides A – J, were isolated from the extract of the fungus. These peptides (structure formulae not shown) are currently undergoing absolute configuration analysis by means of the advanced Marfey’s method and X-ray crystallography. We have already submitted Endolides A - J to a total of 311 individual biological activity assays, in such areas like cytotoxicity, antimicrobial, antiviral, antiplasmodial, psychoactive or anti-inflammatory activity, amongst others.

9. Hydroxylated Sclerosporin Derivatives from the Marine-derived Fungus Cadophora malorum†

Abstract The marine-derived fungus Cadophora malorum was isolated from the green alga Enteromorpha sp. Growth on a biomalt medium supplemented with sea salt yielded an extract from which we have isolated sclerosporin and four new hydroxylated sclerosporin derivatives, namely 15-hydroxysclerosporin (2), 12-hydroxysclerosporin (3), 11-hydroxysclerosporin (4) and 8-hydroxysclerosporin (5). The compounds were evaluated in various biological activity assays. Compound 5 showed a weak fat- accumulation inhibitory activity against 3T3-L1 murine adipocytes.

O 14OH O OH O OH H 10 H H 3 1 6 8 15 5 H H H H OH 12 13 OH 1 2 3

O OH O OH H H

OH H H

OH 4 5

Figure 1. Structure formulae of compounds 1 - 5

Introduction

In recent years, research on the chemistry of marine organisms has experienced a tremendous increase, due to the need for compounds possessing bioactivity with possible pharmaceutical application or other economically useful properties (Anderson et al., 2000). From these organisms, marine fungi are recognized as a valuable source for new and bioactive secondary metabolites that have the potential to lead to innovations in drug therapy (Haefner, 2003). Sclerosporin is a rather rare antifungal and sporogenic cadinane-type sesquiterpene (Katayama et al., 1983; Datta et al., 2007). This substance, initially isolated from Sclerotinia fruticula showed an induction of asexual arthrospore formation in fungal mycelia (Katayama et al., 1983; Kitahara et al., 1984). Interestingly, its (-)-form isolated from Diplocarpon mali did not showed sporogenic activity towards S. frutícula (Kitahara et al., 1984; Sawai et al., 1985).

During our search for new natural products produced from the marine-derived fungus Cadophora malorum, (+)-sclerosporin (1) and four new hydroxylated sclerosporin derivatives (2 – 5) were isolated from a spore culture on agar-BMS media supplemented with artificial sea salt.

Results and Discussion

The molecular formulae of compounds 2 - 5 were deduced by HREIMS to be identical, 13 i.e. C15H22O3, indicating five degrees of unsaturation. The C NMR spectra for all four compounds showed 15 closely similar resonance signals, evidencing that each of the compound belonged to the same structural type. In each case a 13C NMR downfield shifted resonance signal at δ 168 – 170 was found, typical for a carboxylic acid functionality (C-14), whereas a further resonance signal at around δ 70 indicated a hydroxy substituted methylene for compounds 2 and 3, a hydroxy substituted methine for compound 5, and for compound 4 a hydroxylated quaternary carbon (see Tables 1-2, respectively). IR spectroscopic measurements showed a broad absorption band at 3300 cm-1 for 2 - 5, also confirming the presence of a hydroxy substituent in each case. 1 1 H- H COSY data for 2 revealed a spin system ranging from H2-3 via H-1 and H-6 to

H2-9 and to H3-12/H3-13, thus outlining major parts of the planar structure. Protons H2-

15 showed a 1H-1H long range coupling to H-5, which together with an HMBC correlation from H2-15 to C-3 and from H-5 to C-6 completed the first ring of 2. HMBC correlations from H-9 to C-14 connected the carboxylic acid moiety with C-10, which in turn had to be bound to C-1 due to a HMBC correlation from H-1 to C-10 and to C-9. The planar structure of compound 2 was thus that of sclerosporin (1) hydroxylated at C- 15. Sclerosporin itself was also obtained during this study. The molecular formula of 1 was deduced by accurate mass measurement (HREIMS) to be C15H22O2. The final structure of 1 was identified as that of the known compound (+)-sclerosporin by comparing its NMR data and optical rotation with published values (Datta et al., 2007; Kitahara et al., 1984). The carbon skeleton of compounds 3 - 5 and the position of the double bonds were also found to be identical to that of 1, as deduced from 1D and 2D NMR analyses. All compounds are thus cadinane type sesquiterpenes with a sclerosporin nucleus (see Tables 1-2) and differ merely concerning the site of hydroxylation. The position of the hydroxy group in 3 and 4 was established making use of 1H-1H COSY and HMBC data. Thus, in 3 the 13C NMR resonance signal for C-12 (δ 64.9) was shifted downfield, and H-12 (δ 3.52) showed coupling with H-11, placing the hydroxy substituent at C-12. The 13C NMR spectrum of 4 exhibited a downfield shifted quaternary carbon (δ 72.6, C-11) along with singlet proton resonances for the methyl 1 groups CH3-12/CH3-13 in the H NMR spectrum, thus the hydroxy substituent had to be placed at C-11. The 13C NMR spectrum of 5 exhibited only two resonances for methylene groups (C-2, C-3) instead of three such moieties as in 2 - 4. Instead of the methylene group CH2-8 in 2 – 4, in 5 a methine group resonating at δ 68.7 and δ 4.13 in the 13C and 1H NMR spectra respectively was found. The 1H-1H COSY spectrum revealed correlations between H-8 and H-9 as well as H-7, positioning the hydroxy substituent in 5 at C-8. The planar structures of 2 - 5 were thus established to be 15- hydroxysclerosporin (2), 12-hydroxysclerosporin (3), 11-hydroxysclerosporin (4) and 8- hydroxysclerosporin (5).

Table 1. 1H NMR Spectroscopic Data for Compounds 2 – 5.

2 3 4 5 a a b b Position δH (J in Hz) δH (J in Hz) δH (J in Hz) δH (J in Hz) 1 2.58, br d (11.4) 2.58, br d (10.2) 2.54, br d (10.6) 2.43, br d (10.6) 2 a: 1.99, m a: 1.91, m a: 1.89, m a: 1.93, m b: 1.37, qd (11.4, 5.4) b: 1.42, qd (11.7, 5.4) b: 1.50, qd (11.7, 5.5) b: 1.45, qd (11.7, 5.4) 3 2.08, m a: 2.06, m a: 1.98, m a: 2.08, m b: 1.88, m b: 1.95, m b: 1.93, m 5 5.79, d (4.1) 5.52, br s 5.71, br d (5.1) 5.57, br d (4.4) 6 2.04, m 2.10, m 2.11, m 2.10, m 7 1.53, tt (10.2, 5.1) 1.65, tt (10.2, 5.1) 1.68, td (10.3, 5.1) 1.52, ddd (3.2, 8.7, 10.7) 8 a: 2.15, dt (19.8, 5.1) a: 2.27, dt (19.8, 5.1) a: 2.38, dt (19.6, 5.1) 4.13, br d (8.7) b: 1.97, m b: 2.02, m b: 2.01, m 9 7.14, br s 7.10, br s 6.95, br t (3.9) 6.79, d (1.8) 11 2.03, m 2.05, m 2.25, m 12 0.83, d (6.8) 3.77 (dd, 4.8, 10.6) 1.23, s 1.00, d (7.1) 13 0.90, d (6.8) 1.03, d (7.0) 1.18, s 1.10, d (7.1) 14 15 4.03, br s 1.68, br s 1.66, br s 1.68, br s a b CDCl3, 300 MHz. Acetone-d6, 300 MHz.

Table 2. 13C NMR Spectroscopic Data for Compounds 2 – 5.

2 3 4 5 a,c a, c b, c b, c Position δC, mult. δC, mult. δC, mult. δC, mult. 1 34.0, CH 33.8, CH 35.6, CH 35.2, CH

2 25.1, CH2 25.4, CH2 26.2, CH2 26.0, CH2

3 26.2, CH2 30.3, CH2 30.8, CH2 31.5, CH2 4 138.3, qC 135.6, qC 133.7, qC 134.9, qC 5 124.3, CH 123.0, CH 126.6, CH 125.1, CH 6 35.5, CH 35.8, CH 36.9, CH 36.2, CH 7 40.0, CH 38.6, CH 47.2, CH 48.8, CH

8 25.4, CH2 28.0, CH2 29.7, CH2 68.7, CH 9 142.6, CH 142.1, CH 140.2, CH 143.7, CH 10 132.9, qC 133.1, qC 134.3, qC 134.3, qC 11 26.4, CH 35.3, CH 72.6, qC 27.3, CH

12 15.0, CH3 64.9, CH2 27.4, CH3 18.9, CH3

13 21.3, CH3 15.4, CH3 28.7, CH3 21.1, CH3 14 172.2, qC 172.0, qC 168.0, qC 168.0, qC

15 67.3, CH2 23.9, CH3 23.9, CH3 23.9, CH3 a b CDCl3, 75.5 MHz. Acetone-d6, 75.5 MHz. d Implied multiplicities determined by DEPT.

Stereochemistry

The Cotton effect in the CD spectrum of (+)-sclerosporin (1) was similar to that reported in the literature (Kitahara et al., 1985), confirming the absolute configuration of 1 to be that of (+)-sclerosporin (see Table 3). The absolute configurations of compounds 2 - 5 were established by analyses of CD and NOESY spectra. The CD spectra of the hydroxylated compounds 2 - 5 were closely similar to that of (+)- sclerosporin (1), thus the identical absolute configuration can be proposed for compounds 2 – 4 (see Table 3). However, the configuration at position C-11 in 3 remains unresolved. Interestingly, 5 was the only one with a negative specific rotation (see Table 3). This change on specific rotation was most probably caused by the additional stereogenic center at C-8. The configuration at C-8 was established making use of NOESY correlations and 1H-1H coupling constants. A NOESY correlation between H-1 and H-6 is in agreement with the configuration of the other sclerosporin derivatives, and further correlations between H-8 and H-6, H-11, H3-12, H3-13 indicate these protons to be in the same spatial orientation. Furthermore, a 1H-1H coupling constant of 8.7 Hz between H-7 and H-8 indicated a trans-type spatial arrangement. Knowing the absolute configuration of H-1, H-6 and H-7 from CD spectroscopic data, we can thus assign the configuration of 5 to be 1R, 6S, 7R, 8S. We propose the trivial names (+)-15-hydroxysclerosporin, (+)-12-hydroxysclerosporin, (+)-11-hydroxysclerosporin and (-)-8-hydroxysclerosporin for compounds 2, 3, 4 and 5, respectively.

Table 3. Comparison of the CD Spectra and Specific Rotation Values for the Reported Sclerosporins (+ and -) and Compounds 1 - 5.

Compound Δε Maxima and Minima in the CD Optical Rotation Spectra (c 1.6 × 10–6 mol/L, MeOH)

7 a 20 (+) (+)-sclerosporin Δε196 + 11.0 [α] D + 11.4 (c 0.035, MeOH) 5 a 20 (-) (-)-sclerosporin Δε214 + 17.0 [α] D - 11.1 (c 0.090, MeOH) 23 (1) (+)-sclerosporin Δε196 + 10.1, Δε215 - 12.4 [α] D + 14 (c 0.033, MeOH) 23 (1) (+)-sclerosporin [α] D + 57 (c 0.033, CHCl3) 23 (2) (+)-15-hydroxy Δε197 + 5.2, Δε214 - 14.8 [α] D + 64 (c 0.033, CHCl3) 23 (3) (+)-12-hydroxy Δε195 + 6.1, Δε214 - 6.2 [α] D + 69 (c 0.033, CHCl3) 23 (4) (+)-11-hydroxy Δε198 + 3.2, Δε213 - 16.3 [α] D + 73 (c 0.033, CHCl3) 23 (5) (-)-8-hydroxy Δε193 + 8.2, Δε215 - 8.0 [α] D - 62 (c 0.033, CHCl3) aΔε minima for + and – sclerosporin not reported in the literature

Bioactivity assays

Compound 2 was evaluated in the protease elastase (HLE) inhibition assay (tested at 100 µM), in the cytotoxic activity assay against a panel of three cancer cell lines (NCI- H460, MCF7 and SF268, tested at 100 µM), in the protein kinases DYRK1A and CDK5 inhibition activity assay (tested at 10 mM), for inhibition of the viral HIV-1 and HIV-2 induced cytopathogenic effect in MT-4 cells (tested at 50 µg/mL), in the Epstein-Barr virus (EBV) assay (tested at 100 µg/mL), and in the antiplasmodial activity assay against Plasmodium berghei (tested at 25 µM), but did not show any activity. Compound 2 was further tested against a pannel of three respiratory viruses, the severe acute respiratory syndrome coronavirus (SARS) assay, Flu A (H5N1) and Flu B (tested at 100 µg/mL in the three tests), bud did not show any significant activity. Compound 3 was evaluated against two influenza viruses, Flu A (H5N1) and Flu B (tested at 100 µg/mL), but did not show any activity. Compound 5 was evaluated against 3-T3-L1 murine adipocytes assay and showed weak inhibitory activity with an IC50 of 212 µM for triglyceride accumulation inhibition along with an IC50 cytotoxic effect value of 304 µM (see Appendix – bioactivity results). All compounds were tested for sporogenic activity (concentrations from 0.005 µg/mL to 5 µg/mL in BMS-agar plates) but the results were inconclusive, i.e. sporogenesis activity was observed but not with direct correlation with increasing quantity, probably due to compound unstability. All compounds were evaluated in antibacterial (Escherichia coli, Bacillus megaterium),

antifungal (Mycotypha microspora, Eurotium rubrum, and Microbotryum violaceum), and antialgal (Chlorella fusca) assays but did not showed activity at 50 µg/disc dose.

Experimental Section General Experimental Procedures See chapter 3 - General methodology.

Fungal material. See chapter 3 - General methodology.

Culture, extraction and isolation. A sporous culture of Cadophora malorum on a 10 L solid biomalt medium (biomalt 20 g/L, 15 g/L agar) supplemented with sea salt was performed during 3 months. An extraction with 5 L EtOAc yielded 890 mg of extract which was subjected to a VLC fractionation in a silica open column using a gradient solvent system from PE to acetone, namely 10:1, 5:1, 2:1, 1:1, 100 % acetone and 100 % MeOH, yielded a total of 6 fractions. Compounds 1 to 5 were isolated from VLC fraction 2 (112 mg) by HPLC fractionation using PE-acetone 5:1. Sclerosporin (1) was collected in fraction 1 of 7 (2.7 mg, RT 6 min); (+)-15- hydroxysclerosporin (2) was collected in fraction 7 of 7 (14.0 mg, RT 22 min); (+)-12- hydroxysclerosporin (3) was collected in fraction 6 of 7 (7.1 mg) which was further purified by HPLC fractionation with PE-acetone 26:5 (fraction 1 of 2; 4.5 mg, RT 31 min); (+)-11-hydroxysclerosporin (4) was collected in fraction 3 of 7 (2.2 mg, RT 13 min); (-)-8-hydroxysclerosporin (5) was collected in fraction 2 of 7 (3.5 mg, RT 11 min).

23 (1R, 6S, 7R)-(+)-sclerosporin (1): [α] D + 57 (c 0.033, CHCl3); CD (see Table 3);

HREIMS m/z 234.1623 (calcd for C15H22O2, 234.1620); spectroscopic data were identical with the previously reported data.4,5,7

(1R, 6S, 7R)-(+)-15-hydroxysclerosporin (2): yellow amorphous solid (1.4 mg/L, 1.57 23 %); [α] D + 64 (c 0.033, CHCl3); UV (MeOH) λmax (log ε) 217 nm (3.8); CD (see Table -1 1 13 3); IR (ATR) νmax 3397, 2927, 1682 cm ; H NMR and C NMR (see Tables 1 and 2); + LREIMS m/z 250.1; HREIMS m/z 232.1457 [M-H2O] (calcd for C15H20O2, 232.1463).

(1R, 6S, 7S)-(+)-12-hydroxysclerosporin (3): yellow amorphous solid (0.71 mg/L, 23 0.81 %); [α] D + 69 (c 0.033, CHCl3); UV (MeOH) λmax (log ε) 218 nm (3.9); CD (see -1 1 13 Table 3). IR (ATR) νmax 3397, 2923, 1682 cm ; H NMR and C NMR (see Tables 1 + and 2); LREIMS m/z 250.1; HREIMS m/z 232.1463 [M - H2O] (calcd for C15H20O2, m/z 232.1463).

(1R, 6S, 7S)-(+)-11-hydroxysclerosporin (4): yellow amorphous solid (0.22 mg/L, 23 0.25 %); [α] D + 73 (c 0.033, CHCl3); UV (MeOH) λmax (log ε) 219 nm (3.8); CD (see -1 1 13 Table 3); IR (ATR) νmax 3277, 2928, 1681 cm ; H NMR and C NMR (see Tables 1 + and 2); LREIMS m/z 250.1; HREIMS m/z 232.1464 [M - H2O] (calcd for C15H20O2, m/z 232.1463).

(1R, 6S, 7R, 8S)-(-)-8-hydroxysclerosporin (5): yellow amorphous solid (0.35 mg/L, 23 0.39 %); [α] D - 62 (c 0.033, CHCl3); UV (MeOH) λmax (log ε) 220 nm (3.7); CD (see -1 1 13 Table 3); IR (ATR) νmax 3397, 2928, 1688 cm ; H NMR and C NMR (see Tables 1 + and 2); LREIMS m/z 250.1; HREIMS m/z 232.1458 [M - H2O] (calcd for C15H20O2, m/z 232.1463).

Biological Assays. See chapter 3 - General methodology.

10. Novel Bisabolane Sesquiterpenes from the Marine-derived Fungus Verticillium tenerum

Abstract Chemical investigations of the marine-derived fungus Verticillium tenerum yielded two new hydroxylated bisabolane-type sesquiterpenes verticinol A (1) and B (2). The planar structures of the new compounds were elucidated by employing spectroscopic (NMR, UV, and IR) and mass spectrometric techniques. The absolute configuration of the cyclohexenyl moiety was deduced by a combination of CD spectroscopy and NOESY measurements.

15 15 OH OH 3 3 5 6 1 5 6 1 7 7 14 14 9 O 10 O 16 17

HO 11 HO O 12 12 13 13

Figure 1. Structures of compounds 1 and 2

Introduction

Fungi are an extremely valuable source of novel natural products with a wide array of biological activities (König and Wright, 1996). Polyketides are the dominant class of fungal secondary metabolites, however, peptides and heteroaromatic compounds are found in these microorganisms as well (Schneider et al., 2008). Fungal terpenes are known as potent toxins, for example trichothecenes and botrydials, and as plant growth hormones, for example gibberellins and abscisic acid (Pinedo et al., 2008). To date, bisabolane-type sesquiterpenes have been rarely found in fungi. One report refers to -bisabolol produced by a mangrove-derived ascomycete (Laurent et al., 2002), and a second one describes highly oxygenated bicylic bisabolanes from the basidiomycete Cheimonophyllum candidissimum (Stadler et al., 1994). Basidiomycetes of the genus Lepista were found to contain the volatile - and ß-bisabolenes as well as the bicyclic tetrahydrofuran-containing lepistirone (Abraham et al., 1991). Aromatic bisabolanes include curcutetraol, sydonol and waraterpol (Muelhaupt et al., 2005).

In a project focusing on new and bioactive natural products from the marine-derived fungus Verticillium tenerum, which was isolated from an unknown alga and cultivated in a medium supplemented with sea salt, two new bisabolane sesquiterpenes (1, 2) were obtained (Figure 1).

Results and Discussion

The molecular formula of 1 was deduced from accurate MS measurements (HRESIMS) to be C15H26O3, implying three degrees of unsaturation. The IR spectrum showed characteristic absorption bands at 3376 and 1706 cm –1, which indicated the presence of both hydroxy and carbonyl groups. NMR spectra (Table 1) confirmed the presence of 15 carbon atoms with 24 carbon-attached protons, indicating that two protons are associated with hydroxy groups. A more detailed analysis of NMR chemical shifts 3 3 indicated the presence of one sp secondary methyl (H3-14, H = 0.82) and three sp tertiary methyl groups (H3-12, H3-13 and H3-15 with H = 1.37, 1.37 and 1.27). Furthermore, 1H NMR signals for four sp3 methylene groups, two of them aliphatic

(CH2-8 and CH2-9) and a further two alicyclic (CH2-4 and CH2-5), are present, as well 3 as two sp quaternary carbons (C-3, C-11), and the carbonyl carbon C-10 (C = 214.5). There are two downfield shifted 1H NMR signals for methine protons, i.e. the olefinic 1 protons H-1 (H = 5.49) and H-2 (H = 5.63). Two further H NMR signals are slightly 3 downfield shifted and attached to sp hybridized carbons (H-6, H = 2.11 and H-7, H = 1.46). Moreover, the 13C NMR chemical shifts suggested the presence of two tertiary hydroxy groups (C-3, C = 69.7 and C-11, C = 76.1). A significant structural fragment of 1 (fragment A) could be deduced from the 1H-1H COSY spectrum, whereas

15 HO 4 15 H 5 1 CH3 H 7 Fragment A b H 14 8 HO O H 4 5 R a 6 10 Ha HO H Fragment B Hb 12 13 (a) (b)

Figure 2: (a) Fragments of compound 1 as deduced from 1H-1H COSY and 1H-13C HMBC correlations; (b) Key NOESY correlations for compounds 1 and 2 fragment B was delineated from heteronuclear couplings in the 1H-13C HMBC spectrum (Figure 2a). Thus, mutual correlations in the 1H-1H COSY spectrum between the resonances for H-2, H-1, H-6, H2-5 and H2-4 indicated these protons and their respective carbons to be adjacent. In addition, the 1H-1H COSY spectrum revealed a continuous chain of couplings from H3-14 to H-6 and H-9 through H-7 and H-8. The 1 13 correlations between H3-12/H3-13 and both C-10 and C-11 in the H- C HMBC spectrum indicated that methyl groups CH3-12 and CH3-13 are bonded to C-11, which is further connected to C-10 giving rise to fragment B. The connection of fragments A and B was evidenced by an HMBC correlation between H-9 and C-10. Finally, HMBC correlations between H3-15 and C-2, C-3 and C-4 completed the cyclohexenyl ring and thus the planar structure of 1.

Table 1: 1D and 2D NMR spectroscopic data for compound 1.

a,b,e a,b a,b,c a,d a,b,c No. c H (mult., J in COSY HMBC NOESY 1 131.3, CH 5.49, br d (10.3) 2, 5a, 6 2, 3, 5, 6, 7 2, 6, 8a, 8b, 14 2 135.1, CH 5.63, br d (10.3) 1, 4a, 6 1, 4, 6 1, 15 3 69.7, qC

4 38.1, CH2 a: 1.86, m 2, 4b, 5a, 5b 2, 3, 5, 6 4b, 5a, 5b, 15 b: 1.65, m 4a, 5a, 5b 3, 5, 6 4a

5 22.3, CH2 a: 1.67, m 4a, 4b, 5b, 6 4, 6 4a, 5b, 6 b: 1.39, m 4a, 4b,5a, 6 4, 6 4a, 5a 6 40.1, CH 2.11, m 1, 2, 5a, 5b, 7 7 1, 5a, 7, 8a, 9, 14 7 36.3, CH 1.46, m 6, 8a, 8b, 14 14 6, 9, 14

8 27.9, CH2 a: 1.71, m 7, 8b, 9 6, 7 1, 8b, 9 b: 1.44, m 7, 8a, 9 6, 7 8a, 9

9 33.6, CH2 2.55, m 8a, 8b 7, 8, 10 6, 7, 8a, 8b, 12, 13, 14 10 214.5, qC 11 76.1, qC

12 26.5, CH3 1.37, s 10, 11, 13 9

13 26.5, CH3 1.37, s 10, 11, 12 9

14 15.5, CH3 0.82, d (6.6) 7, 8a 6, 7, 8 1, 6, 7, 9

15 28.4, CH3 1.27, s 2, 3, 4 2, 4a a b Chloroform-d1, 300/75 MHz. Assignments are based on extensive 1D and 2D NMR measurements (HMBC, HSQC, COSY). cNumbers refer to proton resonances. dNumbers refer to carbon resonances. eImplied multiplicities determined by DEPT.

The molecular formula of 2 was found to be C17H30O4, as deduced from HRMS and NMR data, implying three degrees of unsaturation. The 13C NMR spectrum showed 17 carbon signals attributed to five methyls, four methylenes, five methines and three quaternary carbons (Table 2). Considering the molecular formula and according to the IR data (3390 cm –1), it was evident that the two remaining protons had to be part of hydroxy groups. The marked similarities in the NMR spectra of 1 and 2 (Tables 1 and 2) suggested that their structures are closely related. Compound 2 is a reduced and acetylated derivative of 1, i.e. the only difference between the molecules is the acetylated hydroxy group at C-10 in 2, compared with the carbonyl group in 1. The connection of the acetyl group (C-16 with C=172.9 and C-17 with C=21.0) to C-10 is supported by a 1H-13C HMBC long range correlation between H-10 and C-16.

Table 2: 1D and 2D NMR spectroscopic data for compound 2.

a,b,e a,b a,b,c a,d a,b,c No. c H (mult., J in COSY HMBC NOESY 1 132.2, CH 5.50, br d (10.3) 2, 5a, 6 2, 3, 5, 6 2, 6, 7, 8a, 8bw, 14 2 136.1, CH 5.62, br d (10.3) 1, 4a, 6 6 1, 15 3 70.2, qC

4 38.7, CH2 a: 1.84, m 5a, 5b 3 4b, 5b, 15 b: 1.70, m 5a, 5b 3 4a

5 23.0, CH2 a: 1.69, m 4a, 4b, 5b, 6 5b, 6 b: 1.43, m 4a, 4b, 5a, 6 4a, 5a 6 41.0, CH 2.17, m 1, 2, 5a, 5b, 7 1, 5a, 7, 14 7 37.9, CH 1.53, m 6, 14 1, 6, 14

8 31.8, CH2 a: 1.39, m 8b, 9a, 9b 7 8b b: 1.17, m 8a, 9a, 9b 7 8a

9 28.5, CH2 a: 1.81, m 8a, 8b, 9b, 10 9b, 10, 12, 13 b: 1.50, m 8a, 8b, 9a, 10 9a 10 81.3, CH 4.81, dd (1.8, 10.3) 9a, 9b 8, 9, 11, 16 9a, 9b, 12, 13, 17 11 72.7, qC

12 25.5, CH3 1.20, s 10, 11, 13 9a, 10, 17

13 26.0, CH3 1.20, s 10, 11, 12 9a, 10, 17

14 16.3, CH3 0.87, d (6.6) 7 6, 7, 8 1, 7, 8a, 8b, 9a

15 28.7, CH3 1.25, s 2, 3, 4 2, 4a, 5b 16 172.9, qC

17 21.0, CH3 2.11, s 16 12, 13 a b Methanol-d4, 300/75 MHz. Assignments are based on extensive 1D and 2D NMR measurements (HMBC, HSQC, COSY). cNumbers refer to proton resonances. dNumbers refer to carbon resonances. eImplied multiplicities determined by DEPT. wweak signal.

HO HO

O O

HO O HO (1) (2)

Figure 2. HMBC correlations of compound 1 and 2 Stereochemistry

The relative configuration at C-3 and C-6 for compounds 1 and 2 was established by means of a NOESY measurement. NOESY correlations were observed between H3-15 and Ha-4, between Ha-4 and both Ha-5 and Hb-5, and between Ha-5 and H-6 (Tables 1 and 2, Figure 2b) indicating methyl group CH3-15 and proton H-6 to be on opposite sides of the molecule. To determine the absolute configuration at C-3 and C-6 of compounds 1 and 2, CD spectra were measured. Compounds 1 and 2 both revealed a negative Cotton effect at around 195 nm. This effect was compared with the Cotton effects of two reference compounds of known configuration, as seen in Figure 3 (spectroscopic description for reference compounds in Mori, 2006) which possess the same cyclohexene ring as compounds 1 and 2, but without the extended side chain (C-7 to C-13). Comparing the CD effects, the absolute configuration of 1 and 2 has to be S at C-3, and hence R at C-6, as it was evident in a relative sense from the NOESY data. The configuration at C-7 for compounds 1 and 2 and at C-10 for compound 2 remains unresolved. Compounds 1 and 2 are new natural products for which the trivial names verticinol A (1) and B (2) are proposed.

a b

c d

Figure 3: CD spectra of compound 1 (a), compound 2 (b), reference compounds (c) and (d). CD values for (c) (c 1.07 x 10-3 mol/L, MeOH)  nm (Δε): 194 (-4.1); CD values for (d) (c 1.07 x 10-3 mol/L, MeOH)  nm (Δε): 193 (+7.5).

Biological activity

Verticinol A was evaluated for cytotoxic activity against three cancer cell lines (NCI- H460, MCF7 and SF268, tested at 100 µM), two bacterial species (Escherichia coli, Bacillus megaterium) and three fungi (Mycotypha microspora, Eurotium rubrum, and Microbotryum violaceum), and the alga Chlorella fusca (at a dose of 50 µg/disc), but did not show any activity. Verticinol A was further evaluated for inhibition of HIV-1 and HIV-2 induced cytopathogenic effects in MT-4 cells (tested at 50 µg/mL), antiplasmodial activity against Plasmodium berghei (tested at 25 µM), and activity against both the Respiratory Syncytial virus (RSV) and the Influenza virus B (Flu B; tested at 100 µg/mL in both assays). However, no activity in any of the assays was found. Verticinol B was evaluated for cytotoxic activity against three cancer cell lines (NCI-H460, MCF7 and SF268, tested at 100 µM), two protein kinases DYRK1A and CDK5 inhibition activity assay (tested at 10 mM), and in the 3T3-L1 murine adipocytes assay (tested at 100 µg/mL). There was no activity in any of these assays.

Experimental section General experimental procedures: See chapter 3 - General methodology.

Fungal material : See chapter 3 - General methodology.

Culture, extraction and isolation: A 10 L culture of Verticillium tenerum grown on solid biomalt medium (biomalt 20 g/L, 15 g/L agar) supplemented with sea salt was maintained during 2 months. An extraction with 5 L EtOAc yielded 980 mg extract which was subjected to a VLC fractionation in a silica open column using a gradient solvent system from PE to acetone, namely 10:1, 5:1, 2:1, 1:1, 100 % acetone and 100 % MeOH, in a total of six fractions. Fraction 3 was separated in a first step by using NP-HPLC with PE – acetone 7:2 to yield 10 subfractions. Subfraction 5 was purified utilizing RP-HPLC with MeOH-H2O 70:30, which afforded the new compound 1 (3.0 mg, R.T. 9 min). Subfraction 9 was purified utilizing RP-HPLC with MeOH-H2O (70:30) yielding subfraction 2, that required further purification utilizing RP-HPLC with

MeOH-H2O (80:20) to yield the new compound 2 (1.6 mg, R.T. 7 min.).

23 Verticinol A (1): Yellowish white oil (0.3 mg/L, 0.31 %); [α] D -74.3 (c 0.23, -1 acetone); UV (MeOH) λmax (log ε): 204 (3.4); IR (ATR): 3376, 2931, 1706 cm ; CD (c 1.97 x 10-3 mol/L, MeOH)  nm (Δε): 194 (-2.0); 1H and 13C NMR data: Table 1; + EIMS: m/z 236 (6), 221 (3), 59 (100); HRMS (ESI, positive mode): m/z (M-H2O) calcd for C15H24O2: 236.1776; found: 236.1776.

23 Verticinol B (2): Light yellow (0.16 mg/L, 0.16 %); [α] D +32.6 (c 0.15, MeOH); UV -1 -3 (MeOH) λmax (log ε): 204 (3.25); IR (ATR): 3390, 2928, 1716 cm ; CD (c 1.68 x 10 mol/L, MeOH)  nm (Δε): 194 (-4.9); 1H and 13C NMR data: Table 2; EIMS: m/z 280 (10), 265 (5), 262 (2), 223 (7), 93 (44), 91 (18), 59 (42), 42 (100); HRMS (ESI, positive + mode): m/z (M-H2O) calcd for C17H28O3: 280.2038; found: 280.2040.

Biological activity: See chapter 3 - General methodology.

11. Discussion

The marine environment is a tremendous source of natural products. Moreover, many of the new compounds isolated from marine micro- and macroorganisms showed prominent biological effects, and some turned out to be valuable sources of lead structures for the development of promising anticancer drug candidates, e.g., the marine-fungal derived lead halimide (Kanoh et al., 1997) or the tunicate-derived compound trabectedin (Rinehart, et al. 1990, Wright, et al. 1990). The main goal of this study was the isolation, identification and biological evaluation of new marine-derived fungal natural products as potential drug leads, from which we report the isolation of 57 secondary metabolites. From these, 25 are new natural products and were isolated from three different fungal strains (further 10 novel N- methylated cyclic peptides are in structure elucidation procedures), confirming once more that endophytic marine-derived fungi are a valuable source of new and interesting secondary natural products.

Considerations on the present future of natural products research

Since the major pharmaceutical companies have terminated or considerably scaled down their natural product drug discovery projects in the 1990s and early 2000s, the academic research in this field is indispensable (Baker et al., 2007). The decline of natural product research in the pharmaceutical industry can be explained by an incompatibility of automated high throughput screening (HTS) programs and the time- consuming work on natural products extract libraries on the one hand, and on the other hand the development of combinatorial chemistry, which generates large so-called screen-friendly synthetic chemical libraries for supporting the HTS efforts (Koehn & Carter, 2005).

The interest of the pharmaceutical industry in some areas of natural products research have been kept or renewed, mainly on cancer research, which still cannot be effectively treated (Hill and Fenical, 2010). The natural products leadfinding success in this area is shown by the fact that 60% of all anticancer agents in clinical use are natural products

or derivatives thereof (Jacob, 2010; Newman and Cragg, 2007; Buttler, 2004). There is currently also a renewed interest from pharmaceutical companies in leadfinding new classes of antibiotics to overcome the growing problem of antibiotic resistance in many bacterial pathogens (Hill and Fenical, 2010; Jacob, 2010). The incredible potential of natural products as leads for new antibiotics is shown by the fact that for bacterial infections, about 80% of the medicines in clinical use are either natural products or derivatives thereof (Jacob, 2010; Newman and Cragg, 2007; Buttler, 2004).

Although we see nowadays a revival of leadfinding marine natural antibiotics, as observed in international natural products conferences, the major research in leadfinding new bioactive natural products is almost reduced to three countries USA, Germany and Japan, and only some isolated institutes spread worldwide. This means that natural products research is still performed by a low number of researchers, taking in consideration the size of pharmaceutical industry and the importance of natural products in society.

A low point in the worldwide research is the low cooperation between institutes isolating new natural products and institutes providing bioassays, as well as the motivation of a natural products chemist in finding bioassays for bioactivity attribution, after the demanding chemical work necessary. This work was heavily complemented with a worldwide cooperation with non-profit institutes and universities for bioassay experiments with agreements performed during research. If it is essential to know where to find valuable natural products, it is equally essential to have bioassays available. An organization connecting bioassay providers and natural product laboratories, where economic interests should be equally distributed according to the amount of work would be thus a solution.

Traditional cultivation and isolation of fungal-derived natural products is indeed time consuming, compound availability is very low and structural complexity can be very high, amongst other disadvantages (intellectual property issues, access to natural chemistry diversity, supply issues, methodological issues, dereplication, etc.) that make it unattractive for pharmaceutical industries, even if the starting biological material has great value. We believe that the rise of natural products research will not depend on funding, but in understanding the biology of microorganisms, which can increase the

rate of isolated new molecules derived from microorganisms, for which a multidisciplinary approach is needed (better than having a chemist, an ecologist, a microbiologist and a pharmacist working together, would be to have one single person with the basic knowledge in all these areas, creating thus a unique vision of nature). This, along with the development of low cost methods will attract funds and researchers to the field.

Furthermore it is necessary to focus on research areas applicable in a short/medium term to the generality of natural product research laboratories worldwide. There has been a great deal of efforts in developing tools for over expressing and discovering bioactive secondary metabolites, heterologous expression in microbial and plant cell cultures, metagenomics or new spectroscopic analytic methods. But low cost methods for the discovery of valuable natural products would lead to the integration of low funded laboratories worldwide in major pharmaceutical discoveries. For example metagenomics is nowadays a hot topic in the field. Its application in cases such as the chemical analysis of gene clusters isolated from unculturable bacteria is indeed state of the art and of unvaluable scientific interest. But these research areas are still (and will be on the next decades) too expensive, time consuming, uncertain regarding success of finding new natural products or in over-expressing pharmaceutically valuable natural products, hence it is promising but far from being a massive tool for academic/industrial natural products discovery. A more interesting approach would be to understand why the great majority of bacterial species are unculturable to this day, i.e. by discovering the biological needs of these bacterial species, one would create a low cost and probably globally applied methodology for the expression of natural products in culture- independent methods. It is considered obviously a huge barrier, but when overcome it would open a new (and applicable massively) world in natural products research.

Natural products laboratories also possess chemical libraries of known but rare compounds isolated in house. Though, many are kept in the freezer, when they should be tested for bioassays as quick as possible, not only because they may possess bioactivity holding the cure for a disease, but because they lose activity during time. Nowadays the field is focusing in leadfinding cytotoxic or antimicrobial new natural products. Though, if a new natural product does not have these biological properties, it

must be seen not as a dead lead, but as the starting point for the remaining immense biological assays available.

The future of natural products research will be considered again economically valuable when the pharmaceutical industry regains the interest on the field. Fast, low cost, and working on biological samples with high probabilities of finding valuable natural products are in need. The potential of other areas not commonly explored in this area should also be investigated, such as searching bioactive proteins from microorganisms. Another poorly understood world but biotechnologically extremely valuable is the study of bacteria associated with eucariotic organisms, mainly because the secondary metabolites produced by these prokaryotes may be the main reason for association/symbiosis, and these kind of associations work as a nature’s pre-clinical trial, having thus these molecules more chances of being used for pharmaceutical applications.

12. Summary

The marine environment is distinguished by unique groups of organisms being the source of a wide array of fascinating structures. Marine microorganisms, such as fungi often occur associated with macroorganisms like algae, sponges or tunicates and produce secondary metabolites with novel structures and potential pharmaceutical significance. The goal of this study was the evaluation of new natural products of marine-derived fungi in order to find novel lead structures for drug development. To achieve this aim seven fungal strains living associated with marine algae and sponges were cultivated during 40 to 60 days, and the extracts tested for bioactivity. Subsequently, the natural products were isolated, their structures determined and their bioactivity established.

From algicolous and sponge-derived fungi, 35 new secondary metabolites were isolated, 14 of which display unprecedented carbon skeletons implying unknown biosynthetic reactions in fungi. All pure compounds were subjected to an extensive panel of bioactivity tests, in a total of around 850 individual biological activity assays against 111 bioactivity targets, from which antiproliferative, antiplasmodial and protease inhibitory properties, as well as affinity to receptors involved in psychoactive activity could be attributed.

The fungus Stachylidium sp. was isolated from the tropical marine sponge Callyspongia cf. C. flammea. This fungus yielded upon cultivation on marine media a plethory of polyketide- (marilines, marilones) and tyrosine-derived metabolites. The skeleton of marilines and marilones is most unusual, and its biosynthesis is suggested to require unusual biochemical reactions in fungal secondary metabolism.

Ten new phthalide derivatives with unusual structural motives, i.e. marilones A – J were

obtained. In the epimeric Figure 1. Structure formulae for marilones A and B compounds marilones I and J the phthalide (=isobenzofuranone) nucleus is modified to an isobenzofuran ring

with ketal and acetal functionality. Marilone A was found to have antiplasmodial activity against Plasmodium berghei with an IC50 of 12.1 µM. Marilone B showed selective antagonistic activity towards the serotonin receptor 5HT2B with a Ki value of 7.7 µM.

Further chemical investigation of this marine-derived fungus led to the isolation of the novel phthalimidine derivatives R R2 1 O marilines A - D. Both enantiomers O N marilines A and B inhibited human O O O leucocyte elastase (HLE) with an IC50 value of 0.86 μM, and (A)R1 =CH3 ; R2 =H acetylcholinesterase (AChE) with

(B)R1 =H; R2 =CH3 IC50 values of 0.18 μM (offset = 49

%) and 0.63 μM (offset = 54 %), Figure 2. Structure formulae of Marilines A and B. respectively.

The diverse secondary metabolism of this Z- E- HO OH fungal strain was additionally demonstrated N N by the identification of yet four further new, putatively tyrosine-derived natural products, i.e. stachylines A – D. The absolute configuration of these novel compounds was O O established by Mosher’s method. Stachyline HO HO A possesses a rare terminal oxime group and

Figure 3. Structure formulae of Stachyline A occurs as an interchangeable mixture of E/Z- isomers.

In the more hydrophilic fractions of the fungal extract, ten N-methylated cyclic peptides, endolides A-J, could be found. The absolute configuration of these compounds and biological activity is currently under investigation.

Further novel compounds, to date with no pharmacological activity attributed, were retrieved from the fungus Cadophora malorum. This green alga derived fungus yielded four new hydroxylated sclerosporin derivatives, namely 15-hydroxysclerosporin, 12- hydroxysclerosporin, 11-hydroxysclerosporin and 8-hydroxysclerosporin, the absolute configuration of which was established by CD spectroscopy. Furthermore, the chemical investigation of Verticillium tenerum yielded the two new hydroxylated bisabolane-type sesquiterpenes verticinol A and B. The absolute configuration of the cyclohexenyl moiety within these metabolites was deduced by a combination of CD spectroscopy and NOESY measurements.

In conclusion, the chemical investigation of marine fungi living associated with algae and sponges led to new natural products, of which the marilines A and B represent the most unusual and unprecedented structural type. Also, this study identified molecules with potent bioactivity, e.g. the HLE and AChE inhibiting activity of marilines A and B.

The results of this study demonstrate that endophytic marine-derived fungi are producers of novel and pharmacologically relevant compounds.

References

Abbenante G. and Fairlie D.P., Med. Chem., 2005, 1, 71-104. Abraham W.R., Hanssen H.P. and Urbasch I., Z. Naturforsch. C, 1991, 46, 169-171. Absalon M.J. and Smith F.O., Expert Opin. Pharmacother., 2009, 10, 1, 57-79. Achenbach H., Mühlenfeld A. and Brillinger G.U., Liebigs Ann. Chem., 1985, 1596-1628. Achilles K. and Bednarski P.J., Biol. Chem., 2003, 384, 817-824. Akazawa T., Miljanich P. and Conn E.E., Plant Physiol., 1960, 35, 535-538. Alvarado J.J., Nemkal A., Sauder J.M., Russell M., Akiyoshi D.E., Shi W., Almo S.C. and Weiss L.M., Mol. Biochem. Parasit., 2009, 168, 2, 158-167. Alves A., Junior J., Slana G., Cardoso J., Wang Q., Lopes R. and Lopes C., Synthetic Comm., 2009, 39, 3693-3709. Amade P., Mallea M. and Bouaïcha, N., J. Antibiot., 1993, 47, 201-207. Anchel M., Hervey A. and Robbins W.J., Proc. Natl. Acad. Sci. U.S.A., 1950, 36, 300-305. Andersen J., Agric. Food Chem., 1987, 35, 1, 60-62. Andersson R.J. and Williams D.E. In Chemistry in Marine Environment; Hester R.E., Harrison R.M. Eds.; The Royal Society of Chemistry: Cambridge, 2000, 55. Andreae M.O., Mar. Chem., 1990, 30, 1-29. Arai K., Yasuji A. and Yamamoto Y., Chem. Pharm. Bull., 1989, 37, 3, 621-625. Aronson J.K., An Account of the Foxglove and Its Medical Uses, 1785-1985, London, New York, Oxford University Press, Oxford 1985. Asami Y., Kakeya H., Okada G., Toi M. and Osada, H., J. Antibiotics., 2006, 59, 724-728. Awad G., Mathieu F., Coppel Y. and Lebrihi A., Can. J. Microbiol., 2005, 51, 59-67. Ayer W.A. and Racok J.S., Can. J. Chem., 1990, 68, 2095-2101. Ayer W.A., Lu P. and Orszanzka H., J. Nat. Prod., 1993, 56, 10, 1835-1838. Baekelandt M., Curr. Opin. Investig. Drugs, 2002, 3, 1517-1526. Baker D.D., Chu M., Oza U. and Rajgarhia V., Nat. Prod. Rep., 2007, 24, 1225-1244. Barnard D.L., Hill C.L., Gage T., Matheson J.E., Huffman J.H., Sidwell R.W., Otto M.I. and Schinazi R.F., Antiviral Res., 1997, 34, 27-37. Barnard D.L., Huffman J.H., Meyerson L.R. and Sidwell R.W., Chemotherapy, 1993, 39, 212- 217. Barrero A.F., Oltra J.E. and Poyatos J.A., Phytochem., 1996, 42, 5, 1427-1433. Bauer A.W., Kirby W.M., Sherris J.C. and Turck M., Am. J. Clin. Pathol., 1966, 45, 4, 493-496. Bedford C.T., Knittel P., Money T., Phillips G.T and Salisbur P., Can. J. Chem., 1973, 51, 694- 697. Bennett J.W. and Chung K-T., Adv. Appl. Microbiol., 2001, 49, 163-184. Bérdy J., J. Antibiotics, 2005, 58, 1-26.

Bernier S.G., Westlin W.F. and Hannig, G., Drugs Future, 2005, 30, 497-508. Bettayeb K., Oumata N., Echalier A., Ferandin Y., Endicott J.A., Galons H. and Meijer L., Oncogene, 2008, 27, 5797-5807. Bhadury P., Mohammad B.T. and Wright P.C., J. Ind. Microbiol. Biotechnol., 2006, 33, 325-337. Bieth J.G. In: Barrett A.J., Rawlings N.D. and Woessner J.F., Handbook of Proteolytic Enzymes, 1998, Academic Press, San Diego Chapter 15. Bingham J.P., Mitsunaga E. and Bergeron Z.L., Chem. Biol. Interact., 2010, 183, 1-18. Birch A.J. and Hussain S.F., J. Chem. Soc., 1969, 1473-1474. Bjarnholt N. and MØller B.L., Phytochemistry, 2008, 69, 1947-1961. Boesenken J., Rec.Trav.Chim (J.R.Neth Chem.Soc), 1939, 53, 528. Bosch F. and Banos J-E., AINE, 1998, 2, 108-117. Bouchillon S., Hoban D., Jordan A., Johnson B., Scangarella N., Shawar R and Johnson J., Retapamulin (SB-275833), a novel pleuromutilin–a multi-center global surveillance of activity against 3,797 beta and alpha hemolytic streptococci isolated from skin and skin structure infections (SSSIs). In: Abstracts and Proceedings of the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), Washington (DC), December 16-19, 2005. Washington (DC): American Society for Microbiology; 2005. p. 109. Abstract F-2061. Brooks G., Burgess W., Colthurst D., Hinks J.D., Hunt E., Pearson M.J., Shea B., Takle A.K., Wilson J.M. and Woodnutt G., Bioorg. Med. Chem., 2001, 9, 1221-1231. Bugni T.S. and Ireland C.M., Nat. Prod. Rep., 2004, 21, 143-163. Bull S.D., Davies S.G., Parkin R.M. and Sánchez-Sancho F., J. Chem. Soc., 1998, 1, 2313-2320. Burton A., The Lancet Infect. Diseas., 2002, 2, 9, 513. Butler M.S., J. Nat. Prod., 2004, 67, 12, 2141-2153 Buttler M.S., Nat. Prod. Rep., 2005, 22, 162-195. Buttler M.S., Nat. Prod. Rep., 2008, 25, 475-516. Calcul L., Inman W.D., Morris A.A., Tenney K., Ratnam J., McKerrow J.H., Valeriote F.A. and Crews P., J. Nat. Prod., 2010, 73, 365-72. Canel C., Moraes R.M., Dayan F.E. and Ferreira D., Phytochemistry, 2000, 54, 115-120. Chen Y.S., Bull. Agr. Chem. Soc. Japan, 1958, 22, 136-142. Chua F. and Laurent G.J., Proc. Am. Thorac. Soc., 2006, 3, 424-427. Chun E., Han C.K., Yoon J.H., Sim T.B., Kim Y.K. and Lee K.Y., Int. J. Cancer., 2005, 114, 124- 130. Cimino G., de Stefano S. and Minale L., Experientia, 1975, 31, 756-757. Crowden R.K. and Ralph B.J., Aust. J. Chem., 1961, 14, 475-478. Cuevas C., Perez M., Martin M.J., Chicharro J.L., Fernandez-Rivas C., Flores M., Francesch A., Gallego P., Zarzuelo M., de la Calle F., Garcia J., Polanco C., Rodriguez I. and Manzanares I., Org. Lett., 2000, 2, 2545-2548.

100

Datta B.K., Rahman M.M., Gray A.I., Nahar L., Hossein S.A., Auzi, A.A., Sarker S.D., J. Nat. Med. 2007, 61, 391-396. Daum R.S., Kar S. and Kirkpatrick P., Nat. Rev. Drug Discov., 2007, 6, 865-866. Deng W-W., Ogita S. and Ashihara H., Phytochem. Lett., 2008, 1, 115-119. Dewick P.M., Medicinal Natural Products: A biosynthetic approach; 3rd ed., John Wiley & Sons Ltd, Chichester, England, 2009, 110-111. Dey D., Pal B.C., Biswas T., Roy S.S., Bandyopadhyay A., Mandal S.K., Giri B.B. and Bhattacharya S., Mol. Cell. Biochem., 2007, 300, 1-2, 149-157. Dickschat J.S., Bode H.B., Wenzel S.C., Müller R. and Schulz S., ChemBioChem, 2005a, 6, 2023- 2033. Dickschat J.S., Helmke E. and Schulz S., Chem. Biodiversity, 2005b, 2, 318-353. Dickschat J.S., Wagner-Döbler I. and Schulz S., J. Chem. Ecol., 2005, 31, 925-947. Dickschat J.S., Wenzel S.C., Bode H.B., Müller R. and Schulz S., ChemBioChem, 2004, 5, 778- 787. Didier P.J., Phillips J.N., Kuebler D.J., Nasr M., Brindley P.J., Stovall M.E., Bowers L.C. and Didier, E.S., Antimicrob. Agents Chemother., 2006, 50, 2146-2155. Dudler R. and Eberl L., Curr. Opin. Biotech., 2006, 17, 268-273. El-Beih A.A., Kato H., Tsukamoto S. and Ohta T., J. Nat. Med., 2007, 61, 2, 175-177. Engels F.K., Sparreboom A., Mathot R.A. and Verweij J., Br. J. Cancer, 2005, 93, 2, 173-177. Esper A.M. and Martin G.S., Expert Opin. Investig. Drugs, 2005, 14, 633-645. Eyberger A.L., Dondapati R. and Porter J.R., J. Nat. Prod., 2006, 69, 1121-1124. Findlay J.A., Li G., Miller J.D. and Womiloju T.O., Can. J. Chem., 2003, 81, 284-292. Flemning, F.F., Nat. Prod. Rep., 1999, 16, 597-606. Fujii I., Ebizuka Y., Chem. Rev., 1997, 97, 2511-2523. Fujita M., Yamada M., Nakajima S., Kawai K.I. and Nagai M., Chem. Pharm. Bull., 1984, 32, 2622-2627. Fumoleau P., Trigo J., Campone M., Baselga J., Sistac F., Gimenez P., Manos L., Fontaine H., Semiond D., Sanderink G., Perard D. and Besenval M., Phase I and pharmacokinetics (PK) study of RPR 116258A given as a weekly 1-hour infusion at day 1, day 8, day 15, day 22 every 5 weeks in patients (pts) with advanced solid tumors.AACR-NCI-EORTC International Conference; October 29-November 2, 2001; Miami Beach, FL. Abstract 282 and poster. Ganesan A., Curr. Opin. Chem. Biol., 2008, 12, 306-317. Garson M.J., Staunton J. and Jones P.G., J. Chem. Soc., 1984, 1021-1026. Gaudilliere J-P., Stud. Hist. Philos. Biol. Biomed. Sci., 2005, 36, 612-644. Gaur S., Newcomb R., Rivnay B., Bell J.R., Yamashiro D., Ramahandran J. and Miljanich, G.P., Neuropharmacol., 1994, 33, 1211-1219.

Gelmon K.A., Latreille J., Tolcher A., Génier L., Fisher B., Forand D., D'Aloisio S., Vernillet

101

L., Daigneault L., Lebecq A., Besenval M. and Eisenhauer E., J. Clin. Oncol., 2000, 18, 24, 4098-4108. Geris R. and Simpson T. J., Nat. Prod. Rep. 2009, 26, 1063-1094. Ghuysen J-M., Int. J. Antimicrob. Agents, 1997, 8, 45-60. Glaser K.B. and Mayer A.M.S., Biochem. Pharmacol., 2009, 78, 440-448. Grimm A., Madduri K., Ali A. and Hutchinson C. R., Gene, 1994, 151, 1-10. Gurib-Fakim A., Mol. Aspects Med., 2006, 27, 1-93. Gyimesi J., Acta Chim. Hung. Tomus, 1965, 45, 323-328. Haefner B., Drug Discov. Today, 2003, 8, 536-544. Hamilton-Miller J.M.T., Int. J. Antimicrob. Agents, 2008, 31, 189-192. Harden E.A., Falshaw R., Carnachan S.M., Kern E.R. and Prichard M.N., Antiviral Res., 2009, 83, 282-289. Harper K., Arif A., Ford E., Strobel G., Porco J., Tomer P., Oneill K., Heidere E. and Grant D., Tetrahedron, 2003, 59, 2471-2476. Hashimoto M., Komori T., Kamiya T., J. Antibiot., 1976, 29, 890-901. Herrero A.B., Martin-Castellanos C., Marco E., Gago F. and Moreno S., Cancer Res., 2006, 66, 8155-8162. Hong, S.W., Han, H.E. and Chae, K.S., J. Liq. Chrom., 1981, 4, 285-292. Horiuchi M., Maoka T., Iwase N. and Ohnishi K., J. Nat. Prod., 2002, 65, 8, 1204-1205. Horiuchi M., Ohnishi K., Iwase N., Nakajima Y., Tounai K., Yamashita M. and Yamada Y., Biosci. Biotechnol. Biochem., 2003, 67, 7, 1580-1583. Hunt E., Drugs Future, 2000, 25, 1163-1168. Hutchinson C.R., Chem. Rev., 1997, 97, 2525. Ignelzi R.J. and Atkinson J.H., Neurosurgery, 1980, 6, 584-590. Ingavat N., Dobereiner J., Wiyakrutta S., Mahidol C., Ruchirawat S. and Kittakoop P., J. Nat. Prod., 2009, 72, 2049-2052. Ingber D., Fujita T., Kishimoto S., Sudo K., Kanamaru T., Brem H. and Folkman J., Nature, 1990, 348, 555-557. Isaaki Y., Matsunaga S., Fusetani, N., Tetrahedron, 1993, 49, 3749-3754. Jacob E.J., Drug Discov. Today, 2010, 15, 11-12, 409-410. Jacobs M.R., Future Microbiol., 2007, 2, 591-600. Ji Y., Bi J-N., Yan B. and Zhu X-D., Chinese Journal of Biotechnology, 2006, 22, 1, 1-6. Jinming G., Lin H. and Jikai L., Steroids, 2001, 66, 771-775. Kanoh K., Kohno S., Asari T., Harada T., Katada J., Muramatsu M., Kawashima H., Sekiya H. and Uno I., Bioorg. Med. Chem. Lett., 1997, 7, 2847-2852. Kanoh K., Kohno S., Katada J., Hayashi Y., Muramatsu M. and Uno I., Biosci. Biotechnol. Biochem., 1999a, 63, 1130-1133. Kanoh K., Kohno S., Katada J., Takahashi J. and Uno I., J. Antibiot., 1999b, 52, 134-141.

102

Kassianidis E., Pearson R.J., Philp D., Org. Lett., 2005, 7, 18, 3833-3836. Katayama M., Marumo S. and Hattori H., Tetrahedron Lett., 1983, 24, 1703-1706. Kawagishi H., Ando M. and Mizuno T., Tetrahedron Lett., 1990, 31, 373-376. Kawahara N., Nozawa K., Nakajima S., Udagawa S. and Kawai K., Chem. Pharm. Bull., 1988, 36, 398-400. Kelly W.L. and Townsend C.A., J. Am. Chem. Soc., 2002, 124, 8186-8187. Kelly W.L. and Townsend C.A., J. Bacteriol., 2005, 187, 739-746. Kerr L.M. and Yoshikami D., Nature, 1984, 308, 282-284. Kim S.-K. and Wijesekara I., J. of Functional Foods, 2010, 2, 1, 1-9. Kim Y.M., An J.J., Jin Y.J., Rhee Y., Cha B.S., Lee H.C. and Lim S.K., J. Mol. Endocrinol., 2007, 38, 455-465. Kitahara T., Kurata H., Matsuoka T., Mori K., Tetrahedron, 1985, 41, 5475-5485. Kitahara T., Matsuoka T., Katayama M., Marumo S. and Mori K., Tetrahedron Lett., 1984, 25, 4685-4688. Klemke C., Kehraus S., Wright A.D. and König G.M., J. Nat. Prod., 2004, 67, 1058-1063. Koehn F.E. and Carter G.T., Nat. Rev. Drug Discov., 2005, 4, 206-220. König G.M. and Wright A.D., Planta Medica, 1996, 62, 193-211. Korba B.E. and Gerin J.L., Antiviral Res. 1992, 19, 55-70. Kruger E.A. and Figg W.D., Expert Opin. Investig. Drugs., 2000, 9, 1383-1396. Kumaki Y., Day C.W., Wandersee M.K., Schow B.P., Madsen J.S., Grant D., Roth J. P., Smee D.F., Blatt L.M. and Barnard D.L., Biochem. Biophys. Res. Commun., 2008, 371, 110- 113. Laurent D., Guella G., Mancini I., Roquebert M.F., Farinole F., Pietra F., Tetrahedron, 2002, 58, 9163-9167. Laustsen G., Shaul M. and Short G., Retapamulin (altabax), Nurse Pract., 2008, 33, 15. Laws P.E., Spark J.I., Cowled P.A. and Fitridge R.A., Eur. J.Vasc. Endovasc, 2004, 27, 1, 6-16. Lee H.S., Choi W.K., Son H.J., Lee S.S., Kim J.K., Ahn S.K., Hong C.I., Min H.K., Kim M. and Myung S.W., Arch. Pharm. Res., 2004, 27, 265-272. Li E., Jiang L., Guo L., Zhang H. and Che Y., Bioorg. & Med. Chem., 2008, 16, 1, 7894-7899. Lin G., Chan S. S-K., Chung H-S. and Li S-L., Studies in Natural Products Chemistry Bioactive Natural Products (Part L), 2005, 32, 12, 2005, 611-669. Lu C., Bai L., Shen Y., Chem. Nat. Compd., 2008, 44, 5, 594-597. Lu H.W., Chong C.R., Hu X. and Liu J.O., J. Med. Chem., 2006, 49, 5645-5648. Marrapodi M. and Chiang J.Y.L., J. Lipid Res., 2000, 41, 514-520. MARTINDALE (database on the internet, www.micromedex.com). Cytarabine, Accessed December 1, 2009. Micromedex Health Care, Martindale-The Complete Drug Reference. MARTINDALE (database on the internet, www.micromedex.com). Vidarabine,. Accessed December 1, 2009. Micromedex Health Care, Martindale-The Complete Drug Reference.

103

Matsumoto K., Nagashima K., Kamigauchi T., Kawamura Y., Yasuda Y., Ishii K., Uotani N., Sato T., Nakai H., Terui Y., Kikuchi J., Ikenisi Y., Yoshida T., Kato T. and Itayaki H., J. Antibiot., 1995, 48, 6, 439-446. Mayer A.M.S., Glaser K.B., Cuevas C., Jacobs R.S., Kem W., Little R.D., McIntosh J.M., Newman D.J., Potts B. C. and Shuster D.E., Trends Pharmacol. Sci., 2010, 31, 6, 255- 265. Mccloskey L., Zonis R., Broskey J., Biswas S., Pizzollo J., Jakielaszek C., Payne D. and Rittenhouse S., In-vitro spectrum of retapamulin activity against organisms associated with skin structure and community acquired infections. In: Abstracts and Proceedings of the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), Washington (DC), December 16-19, 2005. Washington (DC): American Society for Microbiology; 2005. p. 109. Abstract F-2055. McGivern J.G., Drug Discov. Today, 2006, 11, 245-253. McMorris T.C. and Anchel, M., J. Am. Chem. Soc., 1965, 87, 1594-1600. McMorris T.C., Kelner M.J., Wang W., Yu J., Estes L.A. and Taetle R., J. Nat. Prod., 1996, 59, 896-899. McMorris T.C., Yu J., Lira R., Dawe R., MacDonald J.R., Waters S.J. and Kelner M.J., J. Org. Chem., 2001, 66, 6158-6163. Melnikova I. and Golden J., Nat. Rev. Drug Discov., 2004, 3, 993-994. Miljanich G. P., Curr. Med. Chem., 2004, 11, 3029-3040.

Monks A., Scudiero D., Skehan P., Shomaker R., Paull K., Vistica D. and Hose C., J. Natl. Cancer Inst., 1991, 83, 661-757. Moreau A., Lorion M., Couture A., Deniau E. and Grandclaudon P., 2006, J. Org. Chem., 71, 8, 3303-3305. Mori K., Tetrahedron Asymmetry, 2006, 17, 2133-2142. Moulton K.S., Heller E., Konerding, M.A., Flynn E., Palinski W. and Folkman J., Circulation, 1999, 99, 1726-1732. Moya P., Castillo M., Primo-Yúfera E., Couillaud F., Martínez-Máñez R., Garcerá M.-D., Miranda M.A., Primo J. and Martínez-Pardo R., J. Org. Chem., 1997, 62, 8544-8545. Muelhaupt T., Kaspar H., Otto S., Reichert M., Bringmann G. and Lindel T., Eur. J. Org. Chem., 2005, 334-341. Mutschler E., Geisslinger G., Kroemer H.K., Ruth P. and Schäfer-Korting M., Wissenschaftliche Verlagsgesellschaft, Stuttgart, 2008, 419. Nair M.G. and Burke B.A., Phytochem., 1988, 27, 10, 3169-3173. Nair M.G., Burke B.A., Phytochemistry, 1988, 27, 3169-3173. Namikoshi M., Murakami T., Fujiwara T., Nagai H., Niki T., Harigaya E., Watanabe M.F., Oda T., Yamada J. and Tsujimura S., Chem. Res. Toxicol., 2004, 17,12, 1692-1696.

104

Neumann K., Kehraus S., Gütschow M. and König G.M., Nat. Prod. Commun. 2009, 4, 347-354. Newman D.J. and Cragg G.M., J. Nat. Prod., 2007, 70, 461-477. Newman D.J., Cragg G.M. and Battershill C.N., Diving Hyperb. Med., 2009, 39, 216-225. Newman D.J., Cragg G.M. and Snader K.M., Nat. Prod. Rep., 2000, 17, 215-234. Nicholson B., Lloyd G.K., Miller B.R., Palladino M.A., Kiso Y., Hayashi Y. and Neuteboom S.T.C., Anticancer drugs, 2006, 17, 25-31. Nozawa Y., Ito M., Sugawara K., Kazunori H., Mizoue K., J. Antib., 1997, 50, 8, 641-645. Ogawa K., Nakamura M., Hayashi M., Yaginuma S., Yamamoto S., Furihata K., Shin-Ya K. and Seto H., J. Antibiot., 1995, 48, 12, 1396-1400. Olivera B.M., Fusetani, N., ed., Basel, Karger, 2000, 75-85. Pannecouque C., Daelemans D., De Clercq E., Nat. Protocols, 2008, 3, 427-434. Paolucci C. and Rosini G., Tetrahedron: Asymmetry, 2007, 18, 2923-2946. Pham C.T., Nat. Rev. Immunol., 2006, 6, 541.

Pinedo C., Wang C-M., Pradier J-M., Dalmais B., Choquer M., Le Pecheur P., Morgant G., Collado I.G., Cane D.E., Viaud M., ACS Chemical Biology, 2008, 3, 791-801. Pollak F.C. and Berger R.G., Appl. Environ. Microbiol., 1996, 62, 1295-1299. Pommier Y., Kohlhagen G., Bailly C., Waring M., Mazumber A. and Kohn K.W., Biochemistry, 1996, 35, 13303-13309. Prudêncio M., Rodrigues C.D., Ataide R. and Mota M.M., Cell Microbiol., 2008, 10, 218-224. Psychoactive receptors: For experimental details see http://pdsp.med.unc.edu/UNC- CH%20Protocol%20Book.pdf Puri S.C., Verma V., Amna T., Qazi G.N. and Spiteller M., J. Nat. Prod., 2005, 68, 1717-1719. Rauck R.L., Wallace M.S., Burton A.W., Kapural L. and North J.M., Pain Pract., 2009, 9, 327- 337. Rinehart K.L., Holt T.G., Fregeau N.L., Stroh J.G., Kieffer P.A., Sun F., Li L.H. and Martin D.G., J. Org. Chem., 1990, 55, 4512-4515, (Original isolation); (Erratum): Rinehart K.L., Holt T.G., Fregeau N.L., Stroh J.G., Kieffer P.A., Sun F., Li L.H. and Martin D.G., J. Org. Chem., 1991, 56, 1676. Roggo B.E., Petersen F., Sills M., Roesel J.L., Moerker T. and Peter H.H., 1996, J. Antibiot (Tokyo), 49, 1, 13-19. Saroglou V., Karioti A., Demetzos C., Dimas K. and Skaltsa H., J. Nat. Prod., 2005, 68, 1404- 1407. Sasaoka K., Ogawa T., Tsuji H. and Bando N., Biochi. Biophys. Acta, 1980, 630, 137-140. Sashidhara K.V., White K.N. and Crews, P., J. Nat. Prod., 2009, 72, 588-603. Sawai K., Okuno T. and Yoshkawa E., Agric. Biol. Chem., 1985, 49, 2501-2503. Schmitz R. F., Pharm. Hist., 1985, 27, 61-74. Schneider G., Anke H. and Sterner O., Z. Naturforsch C, 1996, 51, 11-12, 802-806.

105

Schneider P., Misiek M. and Hoffmeister D., Mol. Pharm., 2008, 5, 234-242. Schöller E.G., Gürtler H., Pedersen R., Molin S. and Wilkins K., J. Agric. Food Chem., 2002, 50, 2615-2621 Schuh, H., Die Zeit, 2008, 34, 31. Schulte K.E., Ruccker G. and Fachmann H., Tetrahedron Lett., 1968, 4763-4764. Schulz S. and Dickschat J.S., Nat. Prod. Rep., 2007, 24, 814-842. Schulz B., Boyle C., Draeger S., Rommert A.K. and Krohn K., Mycol. Res., 2002, 106, 996-1004. Schulz B., Sucker J., Aust H.-J., Krohn K., Ludewig K., Jones P.G. and Döring D., Mycol. Res., 1995, 99, 1007-1015. Schumacher M., Cerella C., Eifes C., Chateauvieux S., Morceau F., Jaspars M., Dicato M. and Diederich M., Biochem. Pharmacol., 2010, 79, 4, 2010, 610-622. Seale A.P., de Jesus L.A., Kim Se-Y. , Choi Y-H. , Lim H.B., Hwang C-S. and Kim Y-S., Biotechnol. Lett., 2005, 27, 4, 221-225. Seibert S.F., Eguereva E., Krick A., Kehraus S., Voloshina E., Raabe G., Fleischhauer J., Leistner E., Wiese M., Prinz H., Alexandrov K., Janning P., Waldmann H. and König G.M., Org. Biomol. Chem., 2006, 4, 2233-2240.

Seiden M.V., Gordon A.N., Bodurka D.C., Matulonis U.A., Penson R.T., Reed E., Alberts D.S., Weems G., Cullen M. and McGuire W.P., Gynecol. Oncol., 2006, 101, 55-61. Sells M.A., Zelent A.Z., Shvartsman M. and Acs G., J. Virol., 1988, 62, 2836-2844. Sessa C., Cuvier C., Caldiera S., Bauer J., Van den Bosch S., Monnerat C., Semiond D., Pérard D., Lebecq A., Besennval M. and Marty M., Ann Oncol., 2002, 13, 7, 1140-1150. Shimokawa K., Iwase Y., Miwa R., Yamada K. and Uemura D., J. Med. Chem., 2008, 51, 5912- 5914. Shindo K., Yiuji Y., Yukiko O. and Kawai H., J. Antibiot. 1994, 48, 1072-1074. Shiner E.K., Rumbaugh K.P. and Williams S.C., FEMS Microbiol. Rev., 2005, 29, 935-947. Sibbesen O., Koch B., Halkier B.A. and Moller B.L., J. Biol. Chem., 1995, 270, 3506-3511. Sidwell R.W. and Smee D.F., Antiviral Res., 2000, 48, 1-16. Simunovic V., Zapp J., Rachid S., Krug D., Meiser P. and Müller R., 2006, Chembiochem., 7, 8, 1206-1220. Smee D.F., Burger R.A., Warren R.P., Bailey K.W. and Sidwell, R.W., Antiviral Chem. Chemother., 1997, 8, 573-581. Smith G., Biochem. J., 1952, 50, 629. Smith S., J. Chem. Soc., 1930, 508-510. Sperandio V., Trends Immunol., 2004, 25, 505-507. Staats P.S., Yearwood T., Charapata S.G., Presley R.W., Wallace M.S., Byas-Smith M., Fisher R., Bryce D.A., Mangieri E.A., Luther R.R., Mayo M., McGuire D. and Ellis D., J. Am. Med. Assoc., 2004, 291, 63-70.

106

Stadler M., Anke H., Sterner O., Tetrahedron, 1994, 50, 12649-12654. Stevens T., Johnson B., Bouchillon S., Hoban D., Scangarella N., Shawar R., Johnson J. and Badal, R., A multi-center global surveillance study of the in vitro activity of retapamulin (SB-275833), a novel topical pleuromutilin against 2,939 clinical isolates of S. aureus and coagulase negative staphylococci from uncomplicated skin and skin structure infections (SSSIs). In: Abstracts and Proceedings of the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), Washington (DC), December 16-19, 2005. Washington DC: American Society for Microbiology; 2005. p. 109. Abstract F-2062 Stierle A., Hershenhorn J. and Strobel G., Phytochemistry, 1993, 32, 5, 1145-1149. Stierle A., Strobel G. and Stierle D., Science, 1993, 260, 214-216. Stierle A.A., Stierle D.B., Goldstein E., Parker K., Bugni T., Baarson C., Gress J. and Blake D., J. Nat. Prod. 2003, 66, 1097-1100. Strobel G. and Daisy B., Microbiol. Mol. Biol. Rev., 2003, 67, 491-502. Strobel G., Daisy B., Castillo U. and Harper J., J. Nat. Prod., 2004, 67, 257-268. Strobel G.A., Ford E., Worapong J., Harper J.K., Arif A.M., Grant D.M., Fung P. and Chau R.M.W., Phytochemistry, 2002, 60, 179-183. Stuck T.L., Assink B.K., Bates R.C., Erman D.T., Fedij V., Jennings S.M., Lassig J.A., Smith R.J. and Smith T.L., Org. Proc. Res. Dev., 2003, 7, 6, 851-855. Suemitsu R., Ohnishi K., Horiuchi M., Kitaguchi A. and Odamura K., Phytochemistry, 1992, 31, 7, 2325-2326. Suemitsu R., Ohnishi K., Morikawa Y. and Nagamoto S., Phytochemistry, 1995, 38, 495-497. Suzuki E., Sekizaki H. and Inoue S., J. Chem. Soc. Chem. Comm., 1973, 16, 568. Taggart C.C., Greene C.M., Carroll T.P., O’Neill S.J. and McElvaney N.G., Am. J. Respir. Crit. Care Med., 2005, 171, 1070-1076. Takeo T., Phytochemistry, 1974, 13, 1401-1406 Tanaka Y., Sato H., Kageyu A. and Tomita T., J. Chromatogr., 1985, 347, 275-283. Tennant, G. Comprehensive Organic Chemistry, The Synthesis and Reaction of Organic Compounds; Barton, D.; Ollis, W. D.; Eds. Pergamon Press: Oxford, 1979, Vol 2, Chapter 8, 383-590. Thomas X., Expert Opin. Pharmacother., 2009, 10, 221-237. Tsuji H., Bando N., Tadashi O. and Sasaoka K., Biochi. Biophys. Acta, 1981, 677, 326-329 Tsuji H., Ogawa T., Bando N. and Sasaoka K., Biochem Biophys. Res. Comm., 1985b, 130, 2, 633-639. Tsuji H., Ogawa T., Bando N. and Sasaoka K., Biochi. Biophys. Acta, 1985a, 840, 287-290. Turner W.B., Polyketides In Fungal Metabolites, Academic press, London, 1971, 38, 173-175. Tziveleka L-A., Constantinos V. and Vassilios R., Curr. Top. Med. Chem., 2003, 3, 1512-1535. Vane J.R., Nat. New Biol., 1971, 231, 232-235.

107

Vender R.L., J. Invest. Med, 1996, 44, 531-539. Venkateswarlu Y., Reddy N.S., Ramesh P. and Rao J.V., Biochem. Syst. Ecol., 1999, 27, 519-520. Verweij J., J. Clin. Oncol., 2009, 27, 3085-3087. Vrignaud P., Chiron M., Stackhouse M.A., Zuany-Amorim C., Goulaouic H. and Bissery M-C, In vivo synergy between trastuzumab (Herceptin®) and larotaxel (RPR 109881A), a new taxoid. Presented at 98th Annual AACR meeting; April 14-18, 2007; Los Angeles, CA. Abstract 1432. Vrignaud P., Lejeune P. and Bissery M-C, In vivo synergy between doxorubicin, cisplatin or vinorelbine and RPR 109881A, a new taxoid. Poster presented at 96th Annual AACR meeting; April 16-20, 2005; Anaheim, CA. Abstract 3414. Wang J., Li G., Lu H., Zheng Z., Huang Y. and Su W., FEMS Microbiology Letters, 2000, 193, 2, 249-253. Wang Y.X., Gao D., Pettus M., Phillips C. and Bowersox S. S., Pain, 2000, 84, 271-281. Weissman K.J., Bycroft M., Staunton J. and Leadlay P.F., Biochem., 1998, 37, 11012-11017. Wilkins K., Chemosphere, 1996, 32, 1427-1434. Wolfgang K. and Kund K., J. Org. Chem., 1990, 55, 2325-2332. Woynarowska B.A., Woynarowski J.M., Herzig M.C.S., Roberts K., Higdon A.L. and MacDonald J.R., Biochem. Pharmacol., 2000, 59, 1217-1226. Wright A.E., Forleo D.A., Gunawardana G.P., Gunasekera S.P., Koehn F.E. and McConnell O.J., J. Org. Chem., 1990, 55, 4508-4512. Yang X., Strobel G., Stierle A., Hess W.M., Lee J. and Clardy J., Plant Sci., 1994, 102, 1, 1-9. Yap T.A., Carden C.P. and Kaye S.B., Nat. Rev. Cancer, 2009, 9, 167-181. Yoshimoto A., Matzuzawa T., Oki T., Takeuchi T. and Umezawa H., J. Antibiot., 1981, 34, 951. Yue J-M., Chen S-N., Lin Z-W. and Sun H-D., Phytochemistry, 2001, 56, 8, 801-806. Zhan P., Liu X., Fang Z., Pannecouque C. and De Clercq E., Bioorg. Med. Chem., 2009, 17, 6374- 6379. Zhang Y., Yeh J.R., Mara A., Ju R., Hines J.F., Cirone P., Griesbach H.L., Schneider I., Slusarski D.C., Holley S.A. and Crews C.M., Chem. Biol., 2006, 13, 1001-1009. Zikmundova M., Drandarov K., Hesse M., Werner C., Z Naturforsch., 2002, 57C, 660-665.

108

13. APPENDIX 13.1. Bioactivity data Marilines Figure 1. HLE protease inhibition activity assay results for mariline A (W223_A) and

mariline B (W223_B). Calculated IC50values in table 7.

Figure 2. ACEase protease inhibition activity assay results for mariline A (W223_A)

and mariline B (W223_B). Calculated IC50 values in table 7.

109

Table 1. Antiproliferative activity results of mariline A against 5 cancer cell lines

Growth delaying activity (represented by GI50), cytostatic activity (represented by TGI) and cytotoxic activity (represented by LC50) were observed. GI50 is different than the IC50 in that it contains a correction for the starting population. Results in μM.

CEL LINES Mariline A

GI50 29,6 NCI-H460 TGI 60,1 LC5 90,7 GI50 6,9 MCF7 TGI 33,5 LC5 81,0 GI50 25,3 SF268 TGI 56,0 LC5 86,6

GI50 30,8 A549 TGI 73,4 LC5 >100 GI50 29,6 PC3 TGI 80,7 LC5 <100 cancer cell lines: NCI-H460/lung, A549/lung, MCF7/breast and SF268/CNS and CAKI/renal. Averge values in Table 6.

110

Figure 3. Mean graph for antiproliferative activity against 19 cancer cells of mariline B

(GI50s of cell lines are represented as bars vs. the mean of all GI50s (average of all GI50s). Bars to the left denote the sensitive cell lines, those to the right the resistant.)

Table 2. Pairwise coefficient correlations (PCC) of the COMPARE data analysis of mariline B with the synthetic compounds database.

111

Table 3. Pairwise coefficient correlations (PCC) of the COMPARE data analysis of mariline B with the diversity set database.

Table 4. Pairwise coefficient correlations (PCC) of the COMPARE data analysis of mariline B with the marketed drugs database.

112

Table 5. Pairwise coefficient correlations (PCC) of the COMPARE data analysis of mariline B with the standard agents database (Standard agents represent a set of compounds with known mechanism of action. PCC>0.6 gives a first prediction for a putative mechanism of action. PCC is used to measure the similarity degree of the experimental drug (=seed) vs. those of the databases used. Any PCC greater or equal to 0.6 is considered as positive match. The closer to 1 the PCC the greatest the similarity).

113

Figure 4 a-c. Antiplasmodial activity data for marilines A and B Bioactivity test relates to the inhibition of the infection of Plasmodium berghey on the liver stage. The parasite expresses luciferase, thus the infection is measured by luminescence (Bars); the cellular density (i.e. the non-toxicity of the compounds) is measured by fluorescence. The tests were performed in quadruplicate measurements per compound. Samples were dissolved in acetone which was also tested alone. Results show that the solvent has a low effect on the cells of the infection (the X axis of the acetone graphic shows not the concentration of the solvent but the highest quantity of acetone present in the corresponding compound concentrations. Grey bars indicate the non-treated cells results

Figure 4a. Acetone measurements (control)

Acetone

120000 60000 50000 90000 40000 60000 30000 20000 30000 10000 0 0 Luminescence 25 10 5 Non- [] (uM) Treated Alamar blue fluorescence

Figure 4b. Mariline A measurements

Mariline A 120000 60000 50000 90000 40000 60000 30000 20000 30000 10000

Luminescence 0 0

0251051 Alamar bluefluorescence [ ] (uM)

Figure 4c. Mariline B measurements

Mariline B

120000 60000 50000 90000 40000 60000 30000 20000 30000 10000

Luminescence 0 0 025105 1

[ ] (uM) fluorescence blue Alamar

114

Figure 5. Ki determination of mariline B (compound 16587) against the receptor dopamine transporter DAT

(Erratum, value is 5627 nM)

Figure 6. Ki determination of mariline B (compound 16587) against the adregenic receptors Beta 3

Figure 7. Ki determination of mariline B (compound 16587) against the histamine receptor H2.

115

Figure 8. Ki determination for the ligands Mariline C (compound 16594) and D

(compound 16595) against the cannabinoid receptor CB2.

Table 6. Antiproliferative and antiplasmodial biological activity data for marilines A - D Antiproliferative activity against cancer Antiplasmodial activity cell lines (GI50)* (IC50)** Mariline A 24.44 μM 6.68 μM Mariline B 5.00 μM 11.61 μM Mariline C 48.32 μM 13.84 μM Mariline D > 100 μM > 25 μM

*Growth delaying activity represented by GI50 is the average value registered for the tested cell lines; mariline A was tested against 10 cancer cell lines, mariline B against 19 cell lines, marilines C and D against 5 cancer cell lines. All compounds were tested in two independent experiments, each one run in triplicate. Growth rates are the mean of the two experiments. As positive control it was used etoposide treated in the same way. **Antiplasmodial activity test relate to the inhibition of the infection of Plasmodium berghey on the liver stage. The parasite expresses luciferase, thus the infection is measured by luminescence. The tests were performed in quadruplicate measurements per compound.

116

Table 7. Protease HLE and cease inhibition data review for marilines A and B

HLE Porcine Cease equation used for analysis equation used for analysis

v = v0/(1+[I]/IC50) v = (v0-offset)/(1+[I]/IC50)+offset

Mariline A IC50 = 0.86 ± 0.26 μM IC50 = 4.4 ± 1.6 μM IC50 = 0.18 ± 0.08 μM, offset = 49 ± 3%

Mariline B IC50 = 0.86 ± 0.12 μM IC50 = 6.6 ± 1.8 μM IC50 = 0.63 ± 0.20 μM, offset = 54 ± 3% Mariline C > 10 μM > 10 μM > 10 μM Mariline D - - -

IC50 values obtained from quadruplicate measurements at five different inhibitor concentrations.

117

Bioactivity data - Marilones

Figure 9. Antiproliferative activity data for marilones A and C

Growth delaying activity (represented by GI50), cytostatic activity (represented by TGI) and cytotoxic

activity (represented by LC50) were observed. GI50 is different than the IC50 in that it contains a correction for the starting population. Results in μM.

w114 w115

0,1 1 10 100 0,1 1 10 100 150 150

100 NCI-H460 MCF7 SF268 100 NCI-H460 MCF7 SF268

50 50 % Growth Rate % Growth Rate % Growth 0 0

-50 -50

-100 -100 Concentration (μΜ) Concentration (μΜ)

GI50 marilone A GI50 marilone C

CELL LINES (A) (C) Etoposide

GI50 42,9 31,0 0,4 NCI-H460 TGI 80,9 73,8 22,4 LC50 >100 >100 124,2 GI 28,8 20,0 22,8 50 MCF7 TGI 67,7 67,0 118,0

LC50 >100 >100 213,1

GI50 38,4 28,9 7,7 SF268 TGI 70,2 64,2 90,4

LC50 >100 99,6 201,4

Cancer cell lines: NCI-H460/lung, MCF7/breast and SF268/CNS Average values in chapter 4.

118

Figure 10a-c. Antiplasmodial activity data for marilones A and C Bioactivity test relates to the inhibition of the infection of Plasmodium berghey on the liver stage. The parasite expresses luciferase, thus the infection is measured by luminescence (Bars); the cellular density (i.e. the non-toxiciy of the compounds) is measured by fluorescence. The tests were performed in quadruplicate measurements per compound. Samples were dissolved in acetone which was also tested alone. Results show that the solvent has a low effect on the cells of the infection (the X axis of the acetone graphic shows not the concentration of the solvent but the highest quantity of acetone present in the corresponding compound concentrations. Grey bars indicate the non-treated cells results

Figure 10 a. Acetone measurements (control)

Acetone 120000 60000 50000 90000 40000 60000 30000 20000 30000 10000 0 0 Luminescence

25 10 5 Non-Treated fluorescence blue Alamar [] (uM)

Figure 10b. Marilone A

Marilone A 120000 60000 50000 90000 40000 60000 30000 20000 30000 10000 0 0 Alamar blue fluorescence blue Alamar Luminescence 0251051[ ] (μM.)

Figure 10c. Marilone C

Marilone C 120000 60000 50000 90000 40000 60000 30000 20000 30000 10000 0 0 Luminescence 025105 1[ ] (μM.) fluorescence blue Alamar

119

Figure 11. Ki value determination of the antagonistic effect of Marilone B (compound 16591) against the serotonin receptor 5HT2B.

120

13.2. Appendix - Protocols and media Protocol for Laser Microscopy Sample Preparation

Take a small piece of fungi from a petri dish into an eppendorf Take care not to take media I Add 200-400 μL 0.85% NaCl Mix with pipette I Take 10 μL sample and place in PCR-eppendorf I Add 0.5 μL stain solution stock Mix, and incubate in dark for 15 min I Place 5 – 10 μL sample in microscope glass slide I Add 5 μL slow fade reagent enhancer I Check in laser microscope

Material Eppendorfs (normal and PCR-small) Live-dead bac viability kit Slowfade fluorescence enhancer 0.85% NaCl

121

Protocol Advanced Marfey’s Method (Peptide hydrolysis) 1. Take a minimum of 0.5 mg of peptide in a small glass vial with metal screw-top 2. Add 0.5 mL of 6 N HCl 3. Incubate for 16 hours at 110 ºC

4. Dry hydrolysed peptide in a N2 flow until dryness (Marfey reaction) 5. Add 50 μL pure water.

6. Add 40 μL of 1 M NaHCO3 7. Add 100 μl of 1% L-FDLA (or L-FDAA, both are Marfey reagent) (stock solution, 1 mg/100 μL of acetone) 8. Incubate at 70 ºC for 40 minutes (reaction of Marfey reagent and amino acid residues) 9. Cool at room temperature and add 20 μL 2N HCl in order to stop the reaction

10. Dry in N2 flow and add 250 μL ACN for LC-MS analysis (method “Christian 1”; see solvent system in the peptides articles)

Standard amino acids preparation Measure 1 mg of the L-amino acid and in a second vial 0.5 mg of the L-amino acid and 0.5 mg of the D- amino acid. With both vials perform the Marfey reaction above described (point 5. until 10., final concentration for LC-MS 1 mg/ml) Repeat this with every standard amino acids present in the peptide for comparison.

Notes: Hydrolysis is performed to break the peptidic bonds between the amino acid residues. In Marfey’s reaction, the L-L configuration (e. g. L-Phe-L-FDLA) usually comes first as the D-L (e.g. D-Phe-L- FDLA). With both reactions of the standard amino acids we establish first in the mixture of D/L residues the retention time of both D- and L- amino acid configurations reacting with the L-Marfey reagent. Then the vial with only the L-amino acid configuration is basically to confirm which one is the peak corresponding to the product of the reaction L-amino acid with the L-FDLA. You establish then unambiguously the configuration of the residues present in each peak. Then analyse the extracted ion of the same amino acid in your peptide and compare the retention time, it should be the same as L-residue/L- FDLA or D-residue/L-FDLA and you can assign the configuration.

122

Protocol Mosher’s Method for Configurational Determination of Chiral Centers with a Hydroxyl Group/Proton (Secondary and Tertiary alcohols) (a) - Calculations of the mol of Stachymide A in 1 mg 236.26 g ------1 mol 0.001 g ----- X X = 4.233 x 10 -6 mol (b) - Calculation of the mg and final volume of MTPA-Cl (M.W 252.62) corresponding to 10 eq (normally reaction is made with 3 eq). 10 eq = 4.223 x 10-5 mol

252.62 g ----- 1 mol X g ------4.223 x 10-5 mol X = 10.6 mg MTPA-Cl density (ρ) = 1.6, (ρ = m/v) thus vol. of MTPA-Cl to insert is 6.625 μL.

(c) - (R)- and (S)-MTPA esters reaction. Dissolve the secondary alcohol (1 eq, 1 mg, 4.23 x 10-6 µmol) with the corresponding MTPA-Cl (10 eq) in an adequate quantity of deuterated pyridine and DMAP (1 eq) in an NMR tube. The reaction is followed by 1H-NMR to observe the downfield proton shift of H-2’. After 1 hour reaction time (or more if needed), record the final 1H-NMR, dry the sample and re-dissolve in D-acetone for 1H-13C HSQC measurements if necessary. (d) The Δ(S-R) values between (S)- and (R)- MTPA esters can be recorded with 1H-NMR and/or 1H-13C HSQC and they correspond to the difference of the proton shifts near the chiral carbon between the (S)- and (R)- MTPA esters. Normally the reaction is confirmed as successful if both sides have opposite (+ and -) values of Δ(S-R).

Use the general formula in this described figure (positive side in the back, negative in the front, rotate molecule (proton on back side) and determine the R/S configuration.

NOTE: Not even one molecule of water should be present in flasks/pipets/reagents! The 3D model for MPA is the REVERSE of the MTPA, i.e. Δ(R-S), negative Δ in the back, and positive Δ in the front of the model.

123

UV Spectra Calculations UV spectra were recorded on a Perkin-Elmer Lambda 40 with UV WinLab Version 2.80.03 software, using 1.0 cm quartz cells. The molar absorption coefficient ε was determined in accordance with the Lambert-Beer-Law:

ε = A / C.D

A = Absorption at peak maximum (U.A.) C = concentration used in the cuvette (mol/L) D = length of the measurement (normally 1 cm)

Results presented as the logarithm of the molar absorption coefficient (log ε) e.g. “UV

(MeOH) λmax (log Є) 219 nm (log ε 4.11)”

Protocols and Calculations for Circular Dichroism Spectra CD spectra were recorded in MeOH at room temperature using a JASCO J-810-150S spectropolarimeter. The CD (circular dichroism) is measured as ellipticity Θ (in mdeg, millidegrees) and subsequently converted into the molar ellipticity [Θ]M and finally into Δε in accordance to the following equation:

[θ] = θ . M / 10 . C . d

[Θ] = molar ellipticity [degrees x cm2/dmol]

θ = ellipticity (values in mdegrees, which are the Y axis values taken from the CD measurement) M = molecular weight (g/mol) C = concentration in the cuvette (mg/ml ) D = measurement distance, or path lenght (normally 0.1 cm)

Results presented as ε =[θ] / 3300, and written with the molar concentration, c, in the cuvette, e.g. “CD (c 2.8 × 10–4 mol/L, MeOH) λ (ε) = 202 (-9.0), 218 (+12.4), 248 (+6.1)”.

124

Protocol for the effect of (+) sclerosporin and 4 hydroxylated derivatives on sporulation and growth of Cadophora malorum.

5 compounds tested, all under LIGHT conditions Protocol per compound, made in triplicate. sporogenic activity compound concentration to use Spore concentration in 20 ml BMS media growth area number/growth total spore number (μg/mL medium) from STOCK* (cm2) = A area (1 cm2) =B (AxB) 0 0 0.005 1 μL 0.5 10 μL 5 100 μL

*Stock solution: dissolve compounds in acetone (500 μg samples in 500 μL) 9 cm radium petri dish, 20 mL media 1 week waiting time. Spore number count: take 1 cm2 medium, place in a small quantity of water (e.g. 2 mL) and analyze in the microscope with a microscope slide counting chamber. Determine the number of spores and multiply the microscopic volume for the total water volume and subsequently for the total growth area.

125

Media preparations 1L Distilled water Artificial Sea Water (ASW), 1L Minimal Peptone Basal medium KBr 1.0 g DifcoTM Minimal Broth Davis without NaCl 234.8 g Dextrose 10.6 g

MgCl2x6H2O 106.1 g Peptone from meat 3 g

CaCl2x2H2O 14.7 g 1L Distilled water KCl 6.6 g Several carbon sources added from 3g/L

SrCl2x6H2O 0.4 g to 10 g/L, insoluble carbon sources

Na2SO4 39.2 g inoculated with 1% DMSO.

NaHCO3 1.9 g

H3BO3 (boric acid) 0.3 g Peptone medium Peptone 10 g Biomalt medium (BM), 1L NaCl 5 g Biomalt 20 g 1L Distilled water and adjust pH to 7.2 - For BMS, dissolve in ASW -For solid-medium add agar 20 g/L - For BMS-agar, add 15g/L agar Trypticase Soy medium MYA, 1L Pancreatic digest of casein 15 g East extract 4 g Enzymatic digest of soy bean 5 g Malt extract 10 g NaCl 5g Glucose 4 g 1L Distilled water and adjust pH to 7.3 1L Dist. water and adjust to pH 7.3 - For solid-medium, agar 15 g/L - For MYA-ASW dissolve in ASW - For MYA-agar, add 15g/L agar PDA medium, 1L Dissolve 24 g DifcoTM Potato Dextrose Czapek medium, 1L Broth in 1L distilled water. Dissolve 35 g DifcoTM Czapek-dox Broth in 1L distilled water.

MPY medium, 1L Yeast extract 5 g Peptone from Fish 3 g Mannitol 25 g

126

13.3. Appendix - Complete 2D NMR spectroscopic tables Marilines Table 1. 1D and 2D NMR spectroscopic data for marilines A/B

a, b, e a, b a, c a,b, d a, c position δN, δC, mult. δH (J in Hz) COSY HMBC NOESY N 132.4, N 1 166.2, qC 2 116.4, qC 3 157.2, qC 4 119.4, qC 5 161.9, qC 6 101.8, CH 6.95, s 2, 4, 5, 7, 8 8, 9, 1' 7 150.0, qC 8 57.5, CH 4.97, q (6.8) 6, 9 1, 7, 9 6, 9

9 19.4, CH3 1.27, d (6.8) 8 7, 8, N 6, 8 10 62.1, CH3 3.99, s 3 11 11 8.8, CH3 2.12, s 3, 4, 5 10, 9' 12 120.1, qC 13 156.8, qC 14 101.2, CH 6.67, d (2.6) 16 12, 13, 15, 16 18, 1'' 15 160.8, qC 16 105.5, CH 6.56, dd (2.6, 8.6) 14, 17 12, 14, 15 17, 18 17 131.9, CH 7.19, d (8.6) 16 12, 13, 15, N 8, 9, 16

18 55.7, CH3 3.81, brs 15 14, 16 1' 66.2, CH2 a: 4.68, dd (6.6, 12.3) 1'b, 2' 5, 2', 3' 6, 2', 9' b: 4.72, dd (6.6, 12.3) 1'a, 2' 5, 2', 3' 2' 120.6, CH 5.53, t (6.6) 1'a, 1b, 9' 4', 9' 1', 4' 3' 141.5, qC

4' 40.1, CH2 2.12, m 5' 5' 2' 5' 27.0, CH2 2.14, m 4', 6' 4' 6' 6' 124.6, CH 5.10, t (6.6) 5', 8' 5', 8',10' 5', 8' 7' 132.0, qC

8' 25.8, CH3 1.63, brs 6' 6', 7', 10' 6' 9' 16.7, CH3 1.77, brs 2' 2', 3', 4' 1' 10' 17.7, CH3 1.59, brs 6' 6', 7', 8' 1'' 66.1, CH2 a: 4.54, dd (6.6, 12.0) 1''b, 2'', 4'', 5'' 11, 2'', 3'' 12, 2'', 4'', 5'' b: 4.58, dd (6.6, 12.0) 1''a, 2'', 4'', 5'' 11, 2'', 3'' 2'' 120.8, CH 5.35, t (6.6) 1''a, 1''b, 4'', 5'' 4'', 5'' 1'', 5'' 3'' 137.9, qC

4'' 18.2, CH3 1.68, brs 2'' 2'', 3'', 5'' 1'' 5'' 25.7, CH3 1.67, brs 2'' 2'', 3'', 4'' 2'' a b 1 13 acetone-d6, 300/75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments ( H- C, 1H-15N HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT.

9 R R2 6 17 1 O 3' 5' 8' 8 O N 1' 12 1 9' 10' 18 14 3 11 O O O 1'' 10 4''

127

Table 2. 1D and 2D NMR spectroscopic data for mariline C

a, b, e a, b a, c a, d position δC, mult. δH (J in Hz) COSY HMBC 1 167.3, qC 2 116.3, qC 3 156.9, qC 4 119.3, qC 5 161.7, qC 6 101.8, CH 6.92, s 8 2, 3, 4, 5, 7, 8 7 150.0, qC 8 56.9, CH 4.62, q (6.8) 6, 9 1, 2, 6, 7, 9

9 18.9, CH3 1.44, d (6.8) 8 7, 8

10 62.1, CH3 3.98, s 3

11 8.8, CH3 2.09, s 3, 4, 5

12 43.6, CH2 a 3.85, dt (14.0, 5.5) 12b, 13 1, 8, 13 b: 3.33, dt (14.0, 5.5) 12a, 13 1, 8, 13

13 61.7, CH2 3.71, m 12a, 12b 12

1' 66.2, CH2 a: 4.65, dd (6.6, 12.3) 2', 9' 5, 2', 3' b: 4.71, dd (6.6, 12.3) 2' 120.6, CH 5.51, t (6.6) 1', 4', 9' 4' 3' 141.5, qC

4' 40.1, CH2 2.12, m 2', 3', 5', 6'

5' 27.0, CH2 2.14, m 6', 8' 3', 4', 6', 7' 6' 124.6, CH 5.10, t (6.6) 5', 8', 10' 7' 132.1, qC

8' 25.8, CH3 1.63, brs 5', 6' 6', 7', 10'

9' 16.7, CH3 1.76, brs 1', 2' 2', 3', 4'

10' 17.7, CH3 1.58, brs 6' 6', 7', 8' a b 1 acetone-d6, 300/75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments ( H- 13C, 1H-15N HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT. * Weak correlation.

128

Table 3. 1D and 2D NMR spectroscopic data for mariline D

a, b, e a, b a, c a, d position δC, mult. δH (J in Hz) COSY HMBC 1 171.7, qC 2 114.5, qC 3 158.4, qC 4 118.9, qC 5 162.3, qC 6 105.0, CH 6.68, s 2, 3, 4, 5, 8 7 152.1, qC 8 53.2, CH 4.53, q (6.8) 9 1, 2, 6, 7, 9

9 20.8, CH3 1.42, d (6.8) 8 7, 8

10 62.5, CH3 3.95, s 3

11 8.6, CH3 2.15, s 3, 4, 5 aMeOD, 300/75.5 MHz. b Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT.

9 6 8 7 OH HN 5 1 2 3 4 11 O O 10

129

Marilones Table 4. 1D and 2D NMR spectroscopic data for marilone A

a, b, e a, b a, c a, d position δC, mult. δH (J in Hz) COSY HMBC 1 168.2, qC 2 110.0, qC 3 158.0, qC 4 120.4, qC 5 164.2, qC 6 100.8, CH 6.95, s 2, 3, 4, 5, 7, 8 7 154.2, qC 8 77.0, CH 5.43, q (6.6) 11 1, 2, 6, 7, 11

9 62.1, CH3 3.98, s 3

10 8.8, CH3 2.09, s 3, 4, 5

11 20.9, CH3 1.54, d (6.6) 8 7, 8

1' 66.5, CH2 a: 4.69, dd (6.6, 12.1) 1’b, 2' 5, 2', 3' b: 4.74, dd (6.6, 12.1) 1’a, 2’ 5, 2’, 3’ 2' 120.1, CH 5.52, t (6.6) 1'a, 1’b, 9' 1', 4', 9' 3' 142.1, qC

4' 40.1, CH2 2.10, m 5' 2', 6'

5' 26.9, CH2 2.13, m 4', 6' 3', 4', 6', 7' 6' 124.64, CH 5.09, m 4', 5', 8', 10' 4’, 5'; 8’ 7' 132.1, qC

8' 25.8, CH3 1.63, br s 6' 6', 7'

9' 16.7, CH3 1.76, br s 2’ 2', 3', 4'

10' 17.7, CH3 1.58, br s 6' 6', 7' a b CD3COCD3, 300/75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT.

130

Table 5. 1D and 2D NMR spectroscopic data for marilone B

a, b, e a, b a, c a, d position δC, mult. δH (J in Hz) COSY HMBC 1 168.2, qC 2 109.1, qC 3 158.9, qC 4 118.8, qC 5 163.2, qC 6 103.6, CH 6.73, s 2, 3, 4, 5, 8 7 153.9, qC 8 76.6, CH 5.43, q (6.6) 11 1, 2, 6, 7, 11

9 62.0, CH3 3.89, s 3

10 8.6, CH3 2.02, s 3, 4, 5

11 21.0, CH3 1.44, d (6.6) 8 7, 8 a MeOD, 300/75.5 MHz. b Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT.

11 6 OH O 1 O O 9

131

Table 6. 1D and 2D NMR spectroscopic data for marilone C

a, b, e a, b a, c a, d position δC, mult. δH (J in Hz) COSY HMBC

1 68.9, CH2 5.50, s 2, 3, 5, 7, 8 2 128.4, qC 3 153.9, qC 4 124.8, qC 5 159.7, qC 6 102.0, CH 7.03, s 2, 3, 4, 5, 7, 8 7 125.8, qC 8 171.1, qC

9 59.3, CH3 3.96, s 3

10 9.8, CH3 2.15, s 3, 4, 5

1' 66.4, CH2 4.7, d (6.6) 2' 5, 2', 3' 2' 120.5, CH 5.51, t (6.6) 1', 9' 4', 9' 3' 141.6, qC

4' 40.1, CH2 2.10, m 2', 3', 6'

5' 26.9, CH2 2.14, m 4', 6' 3', 4', 6', 7' 6' 124.6, CH 5.10, m 4', 5', 8', 10' 4', 8', 10' 7' 132.05, qC

8' 25.8, CH3 1.63, s (br) 6' 6', 7', 10'

9' 16.7, CH3 1.78, s (br) 2' 2', 3', 4'

10' 17.7, CH3 1.58, s (br) 6' 6', 7', 8' a b CD3COCD3, 300/75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT.

132

Table 7. 1D and 2D NMR spectroscopic data for marilone D

a, b, e a, b a, c a, d position δC, mult. δH (J in Hz) COSY HMBC

1 66.1, CH2 a: 4.39, d (9.9) 1b 2, 3, 7, 9 12 b: 4.41, d (9.9) 1a 2, 3, 7, 9 8 OH 2 119.6, qC HO 3 159.3, qC 1 4 116.1, qC O O 5 157.1, qC 9 6 108.5, CH 6.91, s 2, 3, 5, 7, 8, 11 7 147.2, qC 8 65.9, CH 5.06, q (6.3) 12 2, 6, 7, 12

9 61.8, CH3 3.67, s 3

10 9.1, CH3 2.09, s 3, 4, 5

11 57.8, CH3 3.32, s 1a, 1b

12 25.4, CH3 1.33, d (6.3) 8 7, 8 a b CD3COCD3, 300/75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT.

Table 8. 1D and 2D NMR spectroscopic data for mariline E

a, b, e a, b a, c a, d position δC, mult. δH (J in Hz) COSY HMBC

1 66.9, CH2 4.53, s 2, 3, 7, 9 2 120.3, qC 8 OH 3 160.0, qC HO 4 117.5, qC 1 11 5 157.9, qC O O 9 10 6 111.6, CH 6.77, s 2, 3, 4, 5, 7, 8 7 141.4, qC

8 62.6, CH2 4.64, s 2, 3, 4, 6, 7

9 62.2, CH3 3.74, s 3

10 9.2, CH3 2.15, s 3, 4, 5

11 58.1, CH3 3.42, s 1 a b CD3COCD3, 300/75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT.

133

Table 9. 1D and 2D NMR spectroscopic data for marilone F

a, b, e a, b a, c a, d position δC, mult. δH (J in Hz) COSY HMBC

1 10.1, CH3 0.85, t (7.3) 2 2, 3

2 30.6, CH2 1.35, m 1, 3 1, 3 3 69.4, CH 3.85, m 2, 4 1

4 49.5, CH2 a: 2.41, dd (4.4, 16.5) 3, 4b 2, 3, 5 b: 2.51, m 4a 2, 3, 5 5 214.8, qC 6 48.1, CH 2.87, m 7a, 7b, 16 5, 7, 8, 16

7 29.8, CH2 a: 2.81, m 6, 7b 5, 6, 8, 9, 13, 16 b: 2.53, m 6, 7a 5, 6, 8, 9, 13, 16 8 122.1, qC 9 159.2, qC 10 115.6, qC 11 155.6, qC 12 111.5, CH 6.51, s 8, 10, 11, 14 13 141.9, qC

14 26.3, CH2 2.52, m 15 8, 12, 13, 15

15 15.8, CH3 1.10, t (7.5) 14 13, 14

16 15.9, CH3 0.94, d (6.7) 6 5, 6, 7

17 60.4, CH3 3.65, s 9

18 9.4, CH3 2.08, s 9, 10, 11 a b CD3COCD3, 300/75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT.

134

Table 10. 1D and 2D NMR spectroscopic data for marilone G

a, b, e a, b a, c a, d position δC, mult. δH (J in Hz) COSY HMBC 1 67.1, CH 5.54, dd (1.6, 6.5) 9 2, 3, 7, 8 O 2 139.4, qC 8 OH 3 158.4, qC 9 4 128.1, qC 1 11 HO O 5 159.8, qC 10 6 103.3, CH 6.87, s 2, 4, 5, 8 7 137.2, qC 8 206.4, qC

9 48.4, CH2 a: 3.08, dd (6.5, 19.1) 1, 9b 1, 2, 8 b: 2.50, dd (1.6, 19.1) 1, 9a 1, 2, 8

10 61.1, CH3 4.01, s 3

11 10.0, CH3 2.23, s 3, 4, 5, 6 aMeOD, 300/75.5 MHz. b Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT.

Table 11. 1D and 2D NMR spectroscopic data for marilone H

a, b, e a, b a, c a, d position δC, mult. δH (J in Hz) COSY HMBC 1 166.0, qC 7 2 98.2, qC 6 OH 3 165.6, qC 4 99.1, CH 6.02, s 2, 3, 5, 6 O 1 8 5 164.9, qC O

6 27.1, CH2 2.42, q (7.5) 7 4, 5, 7

7 11.3, CH3 1.13, t (7.5) 6 5, 6

8 8.5, CH3 1.81, s 1, 2, 3 a b CD3COCD3, 300/75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT.

135

Table 12. 1D and 2D NMR spectroscopic data for marilone I

a, b, e a, b a, c a, d a, d position δC, mult. δH (J in Hz) COSY HMBC NOE 1 105.4, CH 6.32, s 2, 3, 6, 7, 8, 13 9, 12, 13 2 121.4, qC 3 154.8, qC 4 119.0, qC 5 160.4, qC 6 100.2, CH 6.60, s 1, 2, 3, 4, 5, 7, 8, 10 10, 11, 12, 1' 7 141.7, qC 8 111.4, qC

9 59.8, CH3 3.90, s 3 1

10 9.3, CH3 2.07, s 2, 3, 4, 5, 6 6

11 28.1, CH3 1.62, s 7, 8 6, 13

12 49.7, CH3 2.85, s 8 1, 6

13 54.2, CH3 3.38, s 1 1, 11

1' 66.1, CH2 4.64, d (6.6) 2', 9' 2', 3', 5' 6, 9' 2' 120.8, CH 5.48, t (6.6) 1', 9' 4', 9' 3' 141.1, qC

4' 40.1, CH2 2.08, m 5' 2', 3', 5', 6', 9' 6'

5' 27.0, CH2 2.11, m 6', 4' 3', 4', 6' 6' 6' 124.6, CH 5.11, m 5', 8', 10' 4', 5', 8' 7' 132.0, qC

8' 25.8, CH3 1.64, br s 6' 6', 7', 10' 6'

9' 16.6, CH3 1.75, br s 2', 1' 2', 3', 4' 1'

10' 17.7, CH3 1.59, br s 6' 6', 7', 8' a b CD3COCD3, 300/75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT.

136

Table 13. 1D and 2D NMR spectroscopic data for marilone J

a, b, e a, b a, c a, d a, d position δC, mult. δH (J in Hz) COSY HMBC NOE 1 104.8, CH 6.02, s 7, 8, 13 9, 11, 13 2 121.5, qC 3 154.9, qC 4 119.2, qC 5 160.4, qC 6 100.2, CH 6.59, s 2, 3, 5, 4, 7, 8, 10 10, 11, 12, 1' 7 141.3, qC 8 110.8, qC

9 59.8, CH3 3.89, s 3 1, 13

10 9.3, CH3 2.06, s 2, 3, 4, 5, 6 6

11 27.8, CH3 1.55, s 7, 8 1, 6, 12

12 50.5, CH3 2.94, s 8 6, 11, 13

13 55.5, CH3 3.50, s 1 1, 9, 12

1' 66.2, CH2 4.63, d (6.6) 2', 9' 5, 2', 3' 6, 9' 2' 120.9, CH 5.48, t (6.6) 1', 9' 4', 9' 3' 141.1, qC

4' 40.1, CH2 2.07, m 5' 2', 3', 5', 6', 9' 6'

5' 27.0, CH2 2.11, m 4', 6' 3', 4', 6' 6' 6' 124.7, CH 5.10, m 5', 8', 10' 4', 5', 8' 7' 132.0, qC

8' 25.8, CH3 1.64, br s 6' 6', 7', 10' 6'

9' 16.7, CH3 1.75, br s 1', 6' 2', 3', 4' 1'

10' 17.7, CH3 1.58, br s 6' 6', 7', 8 a b CD3COCD3, 300/75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT.

137

Stachylines Table 14. 1D and 2D NMR spectroscopic data for stachyline A (Z-).

a,b,e a,b a,b,c a,d a,b,c Position c, mult. H (J in Hz) COSY HMBC NOESY 1 130.1. qC 2 130.5, CH 7.17, d (8.4) 3 4, 6 3, 7 3 115.5, CH 6.88, d (8.4) 2 1, 5 1’a, 1’b, 2 4 158.5, qC 5 115.5, CH 6.88, d (8.4) 6 1, 3 1’a, 1’b, 6 6 130.5, CH 7.17, d (8.4) 5 2, 4 5, 7

7 31.0, CH2 3.60, d (5.5) 8 6, 8 2, 6, 8 8 150.0, CH 6.73, t (5.5) 7 1, 7 7

1’ 72.1, CH2 a: 3.90, dd (7.3, 9.5) 1’b, 2’ 2’, 3’, 4 1’b, 2’, 3, 5 b: 4.01, dd (3.7, 9.5) 1’a, 2’ 2’, 3’, 4 1’a, 2’, 3, 5 2’ 73.9, CH 4.39, m 1’a, 1’b 3’ 146.1, qC

4’ 112.1, CH2 a: 4.89, br s 4’b, 5’ 2’ 4’b b: 5.08, br s 4’a, 5’ 2’, 3’ 4’a

5’ 18.7, CH3 1.78, s 4’a, 4’ b 2’, 3’, 4’ 1’a, 1’b, 2’, 4’a a b CD3COCD3, 300/75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT.

Z- HO N

1 6 5 4 O

138

Table 15. 1D and 2D NMR spectroscopic data for stachyline A (E-)

a,b,e a,b a,b,c a,d a,b,c Position c, mult. H (J in Hz) COSY HMBC NOESY 1 130.1. qC 2 130.5, CH 7.13, d (8.4) 3 4, 6 3, 7 3 115.5, CH 6.88, d (8.4) 2 1, 5 1’a, 1’b, 2 4 158.7, qC 5 115.5, CH 6.88, d (8.4) 6 1, 3 1’a, 1’b, 6 6 130.5, CH 7.13, d (8.4) 5 2, 4 5, 7

7 35.4, CH2 3.39, d (6.2) 8 6, 8 2, 6, 8 8 149.7, CH 7.40, t (6.2) 7 1, 7 7

1’ 72.1, CH2 a: 3.90, dd (7.3, 9.5) 1’b, 2’ 2’, 3’, 4 1’b, 2’, 3, 5 b: 4.01, dd (3.7, 9.5) 1’a, 2’ 2’, 3’, 4 1’a, 2’, 3, 5 2’ 73.9, CH 4.39, m 1’a, 1’b 3’ 146.1, qC

4’ 112.1, CH2 a: 4.89, br s 4’b, 5’ 2’ 4’b b: 5.08, br s 4’a, 5’ 2’, 3’ 4’a

5’ 18.7, CH3 1.78, s 4’a, 4’ b 1’a, 1’b, 2’, 4’a a b CD3COCD3, 300/75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT.

139

Table 16. 1D and 2D NMR spectroscopic data for stachyline B.

a, b, e a, b a, c a, d Position δC, mult. δH (J in Hz) COSY HMBC 1 128.0, qC 2 131.2, CH 7.20, d (8.4) 3 3, 4, 7 3 115.3, CH 6.87, d (8.4) 2 1, 2, 4 4 158.8, qC 5 115.3, CH 6.87, d (8.4) 6 1, 4, 6 6 131.2, CH 7.20, d (8.4) 5 4, 5, 7

7 40.5, CH2 3.51, s 1, 2, 6, 8 8 173.0, qC

1’ 72.1, CH2 a: 4.01, dd (4.0, 9.8) 1’b, 2’ 4, 2’, 3’ b: 3.90, dd (7.2, 9.8) 1’a, 2’ 2’ 73.9, CH 4.40, dd (4.0, 7.2) 1’a, 1’b 1’, 3’, 4’, 5’ 3’ 146.1, qC

4’ 112.1, CH2 a: 4.88, br s 4’a, 5’ 2’, 3’, 5’ b: 5.08, br s 4’b, 5’

5’ 18.8, CH3 1.79, br s 4’a, 4’b 2’, 3’, 4’ a b CD3COCD3, 300/75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT.

140

Table 17. 1D and 2D NMR spectroscopic data for stachyline C

a, b, e a, b a, c a, d Position δC, mult. δH (J in Hz) COSY HMBC 1 132.5, qC 2 130.7, CH 7.13, d (8.4) 3 4, 6, 7 3 115.2, CH 6.84, d (8.4) 2 1, 2, 4, 5 4 158.3, qC 5 115.2, CH 6.84, d (8.4) 6 1, 3, 4, 6 6 130.7, CH 7.13, d (8.4) 5 2, 4, 7

7 39.4, CH2 2.72, t (7.1) 8 1, 2, 6, 8

8 64.1, CH2 3.68, t (7.1) 7 1, 7

1’ 72.1, CH2 a: 4.00, dd (4.3, 9.8) 1’b, 2’ 4, 2’, 3’ b: 3.89, dd (7.2, 9.8) 1’a, 2’ 2’ 74.0, CH 4.39, dd (4.3, 7.2) 1’a, 1’b 1’, 3’, 4’, 5’ 3’ 146.2, qC

4’ 112.0, CH2 a: 4.89, br s 4’b, 5’ 2’, 3’, 5’ b: 5.09, br s 4’a. 5’

4' OH 5' 5 2' 8 1' 1 O OH 7 4 3

141

Table 18 .1D and 2D NMR spectroscopic data for stachyline D

a, b, e a, b a, c a, d Position δC, mult. δH (J in Hz) COSY HMBC 1 127.9, qC 2 131.2, CH 7.19, d (8.4) 3 3, 4, 6, 7 3 115.2, CH 6.87, d (8.4) 2 1, 4, 5 4 159.0, qC 5 115.2, CH 6.87, d (8.4) 6 1, 3, 4 6 131.2, CH 7.19, d (8.4) 5 2, 4, 5, 7

7 40.4, CH2 3.52, br s 1, 2, 6 8 173.2, qC

1’ 71.6, CH2 a: 4.00, dd (4.0, 9.8) 1’b, 2' 4, 2', 3' b: 3.89, dd (6.6, 9.8) 1’b, 2’ 2’ 74.6, CH 3.68, m 1'a, 1’b, 3' 1', 3', 4', 5' 3’ 31.6, CH 1.86, m 2', 4', 5' 1', 2', 5', 4'

4’ 19.4, CH3 0.97, d (6.8) 3' 2', 3', 5'

5’ 17.7, CH3 0.97, d (6.8) 3' 2', 3', 4' a b CD3COCD3, 300/75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT.

142

Sclerosporins Table 19. 1D and 2D NMR Spectroscopic Data for [(+)-15-hydroxysclerosporin]

a, b, e a, b a, c a, d a, c Position δC, mult. δH (J in Hz) COSY HMBC NOESY 1 34.0, CH 2.58, br d (11.4) 2a, 2b, 6 6, 7, 9, 10 6

2 25.1, CH2 a: 1.99, m 1, 2b, 3 1, 3, 6 2b b: 1.37, qd (11.4, 5.4) 1, 2a, 3 1, 3, 6 2a, 7

3 26.2, CH2 2.08, m 2a, 2b 4, 5 4 138.3, qC 5 124.3, CH 5.79, d (4.1) 6, 15 1, 3, 6, 15 6, 12, 15 6 35.5, CH 2.04, m 1, 5, 7 1 1, 8a, 12 7 40.0, CH 1.53, tt (10.2, 5.1) 6, 8a, 8b 6, 11, 12, 13 2b, 11

8 25.4, CH2 a: 2.15, dt (19.8, 5.1) 7, 8b, 9 6, 7, 9, 10, 14 8b, 9 b: 1.97, m 7, 8a, 9 6, 7, 9, 10, 14 8a, 9 9 142.6, CH 7.14, br s 8a, 8b 1, 7, 8, 10, 14 8a, 8b 10 132.9, qC 11 26.4, CH 2.03, m 7, 12, 13 12, 13 12, 13

12 15.0, CH3 0.83, d (6.8) 11 7, 11, 13 5, 11

13 21.3, CH3 0.90, d (6.8) 11 7, 11, 12 11 14 172.2, qC

15 67.3, CH2 4.03, br s 5 3, 4, 5 5 a b CDCl3, 300/75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT.

143

Table 20. 1D and 2D NMR Spectroscopic Data for [(+)-12-hydroxysclerosporin]

a, b, e a, b a, c a, d a, c Position δC, mult δH (J in Hz) COSY HMBC NOESY 1 33.8, CH 2.58, br d (10.2) 2a, 2b, 6 3 6

2 25.4, CH2 a: 1.91, m 1, 2b, 3a, 3b 1, 6 2b b: 1.42, qd (11.7, 5.4) 1, 2a, 3a, 3b 1,6 2a, 7

3 30.3, CH2 a: 2.06, m 2a, 2b, 3b 3b b: 1.88, m 2a, 2b, 3a 2b, 3a 4 135.6, qC 5 123.0, CH 5.52, br s 6, 15 1, 3, 6, 15 6, 12a, 12b, 15 6 35.8, CH 2.10, m 1, 5 8 1, 5 7 38.6, CH 1.65, tt (10.2, 5.1) 6, 8a, 8b 11, 12 2b, 11

8 28.0, CH2 a: 2.27, dt (19.8, 5.1) 7, 8b 6, 7, 9, 10 8b, 9 b: 2.02, m 7, 8a, 9 6, 7, 9, 10 8a, 9 9 142.1, CH 7.10, br s 8a, 8b 1, 7, 8, 10, 14 8a, 8b 10 133.1, qC 11 35.3, CH 2.05, m 7, 12a, 12b, 13 7

12 64.9, CH2 a: 3.77 (dd, 4.8, 10.6) 11, 12b 7, 11, 13 5, 6, 12b b: 3.52, t (10.6) 11, 12a 7, 11, 13 5, 6, 12a

13 15.4, CH3 1.03, d (7.0) 11 7, 11, 12 11 14 172.0, qC

15 23.9, CH3 1.68, br s 5 3, 4, 5 5 a b CDCl3, 300/75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT.

144

Table 21. 1D and 2D NMR Spectroscopic Data for [(+)-11-hydroxysclerosporin]

a, b, e a, b a, c a, d a, c Position δC, mult. δH (J in Hz) COSY HMBC NOESY 1 35.6, CH 2.54, br d (10.6) 2a, 2b, 6 6

2 26.2, CH2 a: 1.89, m 1, 2b, 3a, 3b 1, 6 2b b: 1.50, qd (11.7, 5.5) 1, 2a, 3a, 3b 2a, 7

3 30.8, CH2 a: 1.98, m 2a, 2b b: 1.95, m 2a, 2b 4 133.7, qC 5 126.6, CH 5.71, br d (5.1) 6, 15 1, 3, 6, 15 6, 12, 15 6 36.9, CH 2.11, m 1, 5 5, 7, 8 1, 5, 12 7 47.2, CH 1.68, td (10.3, 5.1) 8a, 8b 6, 11 2b, 8a, 12, 13

8 29.7, CH2 a: 2.38, dt (19.6, 5.1) 7, 8b, 9 6, 7, 9, 10, 11 7, 8b, 9, 13 b: 2.01, m 7, 8a, 9 8a, 9 9 140.2, CH 6.95, br t (3.9) 8a, 8b 1, 7, 8, 10, 14 8a, 8b 10 134.3, qC 11 72.6, qC

12 27.4, CH3 1.23, s 7, 11, 13 5

13 28.7, CH3 1.18, s 7, 11, 12 14 168.0, qC

15 23.9, CH3 1.66, br s 5 3, 4, 5 5 a b Acetone-d6, 300/75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT.

145

Table 22. 1D and 2D NMR Spectroscopic Data for [(-)-8-hydroxysclerosporin]

a, b, e a, b a, c a, d a, c Position δC, mult. δH (J in Hz) COSY HMBC NOESY 1 35.2, CH 2.43, br d (10.6) 2a, 2b, 6 7 6

2 26.0, CH2 a: 1.93, m 1, 2b, 3a, 3b 2b b: 1.45, qd (11.7, 5.4) 1, 2a, 3a, 3b 2a

3 31.5, CH2 a: 2.08, m 2a, 2b, 3b 3b b: 1.93, m 2a, 2b, 3a 3a 4 134.9, qC 5 125.1, CH 5.57, br d (4.4) 6, 15 1, 3, 6, 15 6, 11, 12, 13, 15 6 36.2, CH 2.10, m 1, 5, 7 1, 7, 11 1 7 48.8, CH 1.52, ddd (3.2, 8.7, 10.7) 6, 8, 11 1, 6, 8 12, 13 8 68.7, CH 4.13, br d (8.7) 7, 9 7, 9, 10, 11 6, 9, 11, 12, 13 9 143.7, CH 6.79, d (1.8) 8 1, 7, 10, 14 8 10 134.3, qC 11 27.3, CH 2.25, m 7, 12, 13 7, 8 7, 8, 9, 12, 13

12 18.9, CH3 1.00, d (7.1) 11 7, 11, 13 5, 7, 8, 11

13 21.1, CH3 1.10, d (7.1) 11 7, 11, 12 5, 7, 8, 11 14 168.0, qC

15 23.9, CH3 1.68, br s 5 3, 4, 5 5 a b Acetone-d6, 300/75.5 MHz. Assignments are based on extensive 1D and 2D NMR experiments (HMBC, HSQC, COSY). c Numbers refer to proton resonances. d Numbers refer to carbon resonances. e Implied multiplicities determined by DEPT.

O 14 OH 2 10 3 1 4 6 8 OH 15 5 11 12 13

2D NMR tables for Verticinols in the main text 2D NMR tables for the novel furyl derivative in the main text

146

13.4. Appendix - Spectroscopic data Supporting Information

Note: Supporting information of verticinols A and B, and the furyl derivative 2-(furan-3-yl)acetic acid were integrated in the respective chapters

Supporting Information for Marilines A – D (Stachylidium sp.)

Figure 1. Key HMBC and NOE correlations of marilines A – D

O O N

O O O

NOE Correlations (marilines A and B)

O O N

O O O

HMBC correlations (marilines A and B) (15N HMBC correlations in red arrows)

N HO O O HMBC correlations (marilines C) HMBC correlations (marilines D)

147

Figure 2. HMBC NMR data for marilines A and B

Figure 3. NOE NMR data for marilines A and B

148

Figure 4. Separation chromatogram for marilines A (compound 1) and B (compound 2)

Figure 5. Formation of 2, 4-dihydroxyaniline, putative precursor of marilines A/B

149

Figure 6. Reported structures similar to marilines A-D referred in the marilines chapter

O HO NH

O cichorine O O N OH O HO porritoxin O

N HO OH O

stachybotrin C O OH O

N O

hericenone B

150

Figure 7. Reported natural products with similar backbones related to 2,4- dihydroxyaniline

Fungal endophyte DAOM 221611 from spruce needles: (Findlay et al., 2003) and from Penicillium sp.: (El-Beih et al., 2007)

Porifera Oceanapia sp.: (Venkateswarlu et al., 1999)

Fungus Chaetosphaeria sp.: (Zikmundova et al., 2002)

151

Supporting Information for Marilones A – J ( Stachylidium sp.)

Figure 8. Key HMBC correlations for marilones A - J

152

Table 1. 1H-NMR spectroscopic data comparison of reported values for silvaticol and nidulol with compound 9 (marilones chapter) for the conclusive elucidation as silvaticol

1 H NMR (CDCl3) Position Compound 9 Silvaticol* Nidulol* 1 2 3 4 5 6 7.05 7.04 6.59 7 8 5.39 5.39 5.15 9 3.90 3.91 4.08 10 2.23 2.23 2.19

* (Kawahara et al, 1988)

Table 2. 1H-NMR spectroscopic data comparison of compounds 1 and 3 with silvaticol and nidulol derivatives

1 H NMR (CDCl3) Marilone Marilone 6-(3',3'-dimethylallyloxy-4- 5-(3',3'-dimethylallyloxy-7- position A C methoxy-5-methylphthalide** methoxy-6-methylphthalide** 1 2 3 4 5 6 6.54 7.05 7.08 6.62 7 Protons at C-1/C8 5.39 5.39 5.38 5.18 9 4.04 3.90 3.89 4.03 10 2.18 2.23 2.21 2.15 silvaticol derivative nidulol derivative O O

O O

marilone A O O O

O O marilone C O O O O

O O O

** (Suemitsu et al., 1995) silvaticol derivative nidulol derivative

153

Supporting Information for Stachylines A – D (Stachylidium sp.)

Figure 9a and b. CD spectra of stachylines B and C a) CD spectra of stachyline B

b) CD spectra of stachyline C

Figure 10. Key HMBC correlations of stachylines A - D

HOOC O OH

(A) (B)

HO O OH

154

Figure 11. 1H NMR spectroscopic data of the respective (R)- and (S)-MTPA esters of stachyline B.

H3-5’ H-3’

H2-1’

H3-5’

H- H2-1’

Figure 12 (a-c). 1H-13C HSQC spectroscopic data of stachyline B and the respective (R)- and (S)-MPTA esters. a) HSQC spectrum of stachyline B:

155

b) HSQC spectrum of S- Mosher

c) HSQC spectrum of R- Mosher

156

Figure 13. LC-MS (ESI, 2 experiments in + mode) data for stachyline A (E- and Z-)

Peak 16.05 min., 236.4 and 236.3 (M+H)+ Peak 16.61 min., 236.1 and 236.3 (M+H)+

TWC of DAD Spectral Data: from Sample 6 (VLC 3.3.2) of 2009-11-20.wiff Max. 4,4e4 mAU.

16,05 16,61 4,0e4 3,0e4 2,0e4 1,0e4 23,56 0,0 Absorbance, mAU Absorbance,

4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time, min +Q1: Exp 1, 16,091 to 16,218 min from Sample 6 (VLC 3.3.2)... Max. 4,0e5 cps. +Q1: Exp 2, 16,611 to 16,734 min from Sample 6 (VLC 3.3.2)... Max. 1,3e5 cps.

236,1 3,8e5 8,0e4 3,0e5 6,0e4 253,1 2,0e5 236,4 4,0e4

Intensity, cps 1,0e5 Intensity, cps 258,5 295,1 218,3 253,9 258,3 274,0 2,0e4 235,4 252,8 276,4 210 220 230 240 250 260 270 280 290 200 210 220 230 240 250 260 270 280 290 m/z, amu m/z, amu +Q1: Exp 2, 16,101 to 16,227 min from Sample 6 (VLC 3.3.2)... Max. 1,0e5 cps. +Q1: Exp 1, 16,601 to 16,724 min from Sample 6 (VLC 3.3.2)... Max. 3,1e5 cps.

236,3 1,00e5 253,1 3,0e5 218,4 8,00e4 2,5e5 230,8 2,0e5 6,00e4 258,5 1,5e5 236,3 4,00e4 1,0e5 247,1 253,1 Intensity, cps 219,3 Intensity, cps 2,00e4 253,9 258,4 5,0e4 237,1 295,3 261,6 218,6 238,3 250,6 210 220 230 240 250 260 270 280 290 210 220 230 240 250 260 270 280 290 m/z, amu m/z, amu

157

Supporting Information for Hydroxylated Sclerosporin Derivatives (Cadophora malorum)

Figure 14. Infra-red spectra of sclerosporin and the four novel hydroxylated sclerosporin derivatives

Figure 15. Key HMBC correlations for the four novel hydroxylated sclerosporin derivatives

(+)-15-hydroxysclerosporin (2) (+)-12-hydroxysclerosporin (3) (+)-11-hydroxysclerosporin (4) (-)-8-hydroxysclerosporin (5)

Figure 16. Key NOESY correlations for four novel hydroxylated sclerosporin derivatives

(+)-15-hydroxysclerosporin (2) (+)-12-hydroxysclerosporin (3) (+)-11-hydroxysclerosporin (4) (-)-8-hydroxysclerosporin (5)

158

Figures 17 a-e. CD spectra for sclerosporin and the hydroxylated sclerosporin derivatives. Respective absolute configuration on the right side. a) (+)-15-hydroxysclerosporin

b) (+)-12-hydroxysclerosporin

(-)-8-Hydroxy

c) (+)-11-hydroxysclerosporin

d) (-)-8-hydroxysclerosporin

e) (+)-sclerosporin

O OH H

J =4.9Hz J = 10.7 Hz

159

13.5. 1H and 13C NMR spectra

13.5.1. 1H and 13C NMR spectra of new molecules

New molecules from Stachylidium sp.

New sclerosporing derivatives from Cadophora malorum

New molecules from Verticillium tenerum

160

1 H NMR spectra (300 MHz, CD3COCD3) of marilines A and B

R2 R1 O O N

O O O

13 C NMR spectra (75 MHz, CD3COCD3) of marilines A and B

161

1 H NMR spectrum (300 MHz, CD3COCD3) of mariline C

13 C NMR spectrum (75 MHz, CD3COCD3) of mariline C

162

1H NMR spectrum (300 MHz, MeOD) of mariline D

13C NMR spectrum (75 MHz, MeOD) of mariline D

163

1 H NMR (300 MHz, CD3COCD3) of marilone A

13 C NMR (75 MHz, CD3COCD3) of marilone A

164

1 H NMR (300 MHz, CD3COCD3) of marilone B

13 C NMR (75 MHz, CD3COCD3) of marilone B

165

1 H NMR (300 MHz, CD3COCD3) of marilone C

13 C NMR (75 MHz, CD3COCD3) of marilone C

166

1 H NMR (300 MHz, CD3COCD3) of marilone D

13 C NMR (75 MHz, CD3COCD3) of marilone D

167

1H NMR (300 MHz, MeOD) of marilone E

13C NMR (75 MHz, MeOD) of marilone E

168

1 H NMR (300 MHz, CD3COCD3) of marilone F

13 C NMR (300 MHz, CD3COCD3) of marilone F

169

1H NMR (300 MHz, MeOD) of marilone G

13C NMR (75 MHz, MeOD) of marilone G

170

1 H NMR (300 MHz, CD3COCD3) of marilone H

13 C NMR (75 MHz, CD3COCD3) of marilone H

171

1 H NMR (300 MHz, CD3COCD3) of marilone I

13 C NMR (75 MHz, CD3COCD3) of marilone I

172

1 H NMR (300 MHz, CD3COCD3) of marilone J

13 C NMR (75 MHz, CD3COCD3) of marilone J

173

1 H NMR (300 MHz, CD3COCD3) of stachyline A H-5`(E- and Z-)

H-3,5 (E- and Z-) H-7 (Z-) H-7 (E-)

H-8 (E-) H-8 (Z-) H-2,6 H-4`(E-and Z-)

H-2`

13 C NMR (75 MHz, CD3COCD3) of stachyline A

174

1 H NMR (300 MHz, CD3COCD3) of stachyline B

13 C NMR (75 MHz, CD3COCD3) of stachyline B

175

1 H NMR (300 MHz, CD3COCD3) of stachyline C

HOH2C O OH

13 C NMR (75 MHz, CD3COCD3) of stachyline C

176

1 H NMR (300 MHz, CD3COCD3) of stachyline D

13 C NMR (75 MHz, CD3COCD3) of stachyline D

177

1 H NMR (300 MHz, CD3COCD3) of 2-(furan-3-yl)acetic acid

13 C NMR (75 MHz, CD3COCD3) of 2-(furan-3-yl)acetic acid

178

1 H NMR spectrum (300 MHz, CDCl3) of 15-hydroxysclerosporin

13 C NMR spectrum (75 MHz, CDCl3) of 15-hydroxysclerosporin

179

1 H NMR spectrum (300 MHz, CDCl3) of 12-hydroxysclerosporin

13 C NMR spectrum (75 MHz, CDCl3) of 12-hydroxysclerosporin

180

1 H NMR spectrum (300 MHz, acetone-d6) of 11-hydroxysclerosporin

13C NMR spectrum (75 MHz, acetone-d6) of 11-hydroxysclerosporin

181

1 H NMR spectrum (300 MHz, acetone-d6) of 8-hydroxysclerosporin

13 C NMR spectrum (75 MHz, acetone-d6) of 8-hydroxysclerosporin

182

1 H NMR (300 MHz, CDCl3) of verticinol A

13 C NMR (75 MHz, CDCl3) of verticinol A

183

1H NMR (300 MHz, MeOD) of verticinol B

13C NMR (75 MHz, MeOD) of verticinol B

184

13.5.2. 1H and 13C NMR spectra of known molecules

(trivial names in the 1D NMR spectra) Eurotium sp.

(1) (2) (3) 1 - 3. (Arai et al., 1989) Phialophorophoma litoralis

(4) (5) (6) 4. (Garson et al., 1984) 5 - 6. (Klemke et al., 2004)

Cadophora Malorum Verticillium cinnabarinum Verticillium tenerum

(7) (8) (9) 7. (Kitahara et al., 1984) 8. (Bull et al., 1998) 9. (Boeesenken, 1939) Acremonium sp

(10) (11) (12)

10. (Gyimesi, 1965) 11. artefact derived from 10. 12. (Schulte et al., 1968; Jinming et al., 2001)

185

Stachyllidium sp.

O O

(13) (14) (15) (16) (17)

(18) (19) (20) (21) (22)

(23) (24) (25) (26)

13. (Andersen, 1987; Alves et al., 2009). 14. (Ayer et al., 1993) 15. (Schneider et al., 1996) 16. (Yue et al., 2001) 17. (Kawahara et al., 1988) 18. (Schneider et al., 1996) 19. (Awad et al., 2005). 20. (Chen, 1958; Crowden and Ralph, 1961) 21. (Nair and Burke, 1988) 22. (Barrero et al., 1996) 23. (Jinming et al., 2001) 24. (Hong, 1981) 25. (Turner, 1971) 26. (Lu et al., 2008).

186

Compound 1, (tetrahidroauroglaucin/aspergin), Eurotium sp., D-acetone 1H NMR

13C NMR

187

Compound 2, (flavoglaucin), Eurotium sp., CD3COCD3. 1H NMR

13C NMR

188

Compound 3, (isotetrahydroauroglaucin), Eurotium sp., CD3COCD3. 1H NMR

13C NMR

189

Compound 4, (R-mellein), Phialophorophoma litoralis, CDCl3. 1H NMR

13C NMR

190

Compound 5, [(-)-hexylitaconic acid], Phialophorophoma litoralis, CDCl3. 1H NMR

OH OH

O Hexylitaconic acid Chemical Formula: C11H18O4 Exact Mass: 214,1205 Molecular Weight: 214,2582

13C NMR

191

Compound 6, [(-)-9-hydroxy Hexylitaconic acid 1-half-methyl ester],

Phialophorophoma litoralis, CDCl3. 1H NMR

13C NMR

192

Compound 7, [(+)-sclerosporin], Cadophora malorum, CDCl3. 1H NMR

13C NMR

193

Compound 8, (cyclo-proline-isoleucine), Verticillium cinnabarinum, MeOD 1H NMR

13C NMR

194

Compound 9, (3, 4-dimethoxy phenol) , Verticillium tenerum , CDCl3. 1H NMR

13C NMR

195

Compound 10, (crotocin), Acremonium sp., CDCl3. 1H NMR

13C NMR

196

Compound 11, (probable Crotocin artefact - open epoxyde), Acremonium sp., MeOD. 1H NMR

13C NMR

197

Compound 12, (ergosta-4,6,8 (14), 22-tetraen-3-one, impure sample), Acremonium sp.,

CDCl3. 1H NMR

13C NMR

198

Compound 13,(1, 2, 4-trimethoxy-benzene), Stachyllidium sp.,CDCl3. 1H NMR

13C NMR

199

Compound 14 (isosclerone), Stachyllidium sp.,CDCl3. 1H NMR

13C NMR

200

Compound 15, (tirosol), Stachyllidium sp.,CDCl3. (H-NMR in MeOD, C-NMR in D- acetone) 1H NMR

13C NMR

201

Compound 16, (sterol), Stachyllidium sp., D-acetone 1H NMR

13C NMR

202

Compound 17, (nidulol), Stachylidium sp., D-acetone 1H NMR

13C NMR

203

Compound (18) (furyl derivative) Stachylidium sp., MeoD 1H NMR

13C NMR

204

Compound (19), (tyrosine derivative), Stachylidium sp., MeoD 1H NMR

13C NMR

205

Compound 20, para-hydroxyphenylacetic acid, Stachylidium sp., MeoD 1H NMR

13C NMR

206

Compound 21, phenylacetic acid, Stachylidium sp., D-acetone 1H NMR

13C NMR

207

Compound 22, benzoic acid, Stachylidium sp., D-acetone 1H NMR

13C NMR

208

Compound 23, sterol, Stachylidium sp., D-acetone 1H NMR

13C NMR

209

Compound 24, (2-furoic acid), Stachylidium sp., MeoD 1H NMR

No 13C NMR ; m/z 112.02; LR-LC-MS, found 110.8 (M–H)+ The assignment of NMR shifts was supported using the ACD NMR predictor software (ACD/Labs®).

210

Compound 25, (3,4-dihydroxybenzoic acid/Catechuic acid), Stachylidium sp., MeoD 1H NMR

13C NMR

211

Compound 26, (-(furan-2-yl)ethane-1,2-diol ), Stachylidium sp., MeoD 1H NMR

13C NMR

212